Zcytor19 heterodimeric cytokine receptor polynucleotides, polypeptides, antibodies and methods

ABSTRACT

Novel methods are disclosed for forming a heterodimeric receptor complex with IL-28R and CRF2-4. The methods may be used for detecting and treating viral infections in in vitro and in vivo. Ligand-binding receptor polypeptides can also be used to block ligand activity in vitro and in vivo. The present invention also includes methods for producing the protein, uses therefor and antibodies thereto.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/723,364, filed Mar. 12, 2010, which is a divisional of U.S.application Ser. No. 11/539,780, filed Oct. 9, 2006, which is acontinuation of U.S. application Ser. No. 10/420,034, filed Apr. 18,2003, now abandoned, which claims the benefit of U.S. ProvisionalApplication Ser. No. 60/373,813, filed Apr. 19, 2002, all of which areherein incorporated by reference.

BACKGROUND OF THE INVENTION

Hormones and polypeptide growth factors control proliferation anddifferentiation of cells of multicellular organisms. These diffusablemolecules allow cells to communicate with each other and act in concertto form cells and organs, and to repair damaged tissue. Examples ofhormones and growth factors include the steroid hormones (e.g. estrogen,testosterone), parathyroid hormone, follicle stimulating hormone, theinterleukins, platelet derived growth factor (PDGF), epidermal growthfactor (EGF), granulocyte-macrophage colony stimulating factor (GM-CSF),erythropoietin (EPO) and calcitonin.

Hormones and growth factors influence cellular metabolism by binding toreceptors. Receptors may be integral membrane proteins that are linkedto signaling pathways within the cell, such as second messenger systems.Other classes of receptors are soluble molecules, such as thetranscription factors. Of particular interest are receptors forcytokines, molecules that promote the proliferation and/ordifferentiation of cells. Examples of cytokines include erythropoietin(EPO), which stimulates the development of red blood cells;thrombopoietin (TPO), which stimulates development of cells of themegakaryocyte lineage; and granulocyte-colony stimulating factor(G-CSF), which stimulates development of neutrophils. These cytokinesare useful in restoring normal blood cell levels in patients sufferingfrom anemia, thrombocytopenia, and neutropenia or receiving chemotherapyfor cancer.

The demonstrated in vivo activities of these cytokines illustrate theenormous clinical potential of, and need for, other cytokines, cytokineagonists, and cytokine antagonists. The present invention addressesthese needs by providing new a hematopoietic cytokine receptor, as wellas related compositions and methods.

The present invention provides such polypeptides for these and otheruses that should be apparent to those skilled in the art from theteachings herein. These and other aspects of the invention will becomeevident upon reference to the following detailed description of theinvention.

DESCRIPTION OF THE INVENTION

Prior to setting forth the invention in detail, it may be helpful to theunderstanding thereof to define the following terms:

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985;Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase(Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985),substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204-10, 1988),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2: 95-107, 1991. DNAs encoding affinity tags are availablefrom commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

The term “allelic variant” is used herein to denote any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity of<10⁹ M⁻¹.

The term “complements of a polynucleotide molecule” is a polynucleotidemolecule having a complementary base sequence and reverse orientation ascompared to a reference sequence. For example, the sequence 5′ ATGCACGGG3′ is complementary to 5′ CCCGTGCAT 3′.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

The term “expression vector” is used to denote a DNA molecule, linear orcircular, that comprises a segment encoding a polypeptide of interestoperably linked to additional segments that provide for itstranscription. Such additional segments include promoter and terminatorsequences, and may also include one or more origins of replication, oneor more selectable markers, an enhancer, a polyadenylation signal, etc.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

The term “isolated”, when applied to a polynucleotide, denotes that thepolynucleotide has been removed from its natural genetic milieu and isthus free of other extraneous or unwanted coding sequences, and is in aform suitable for use within genetically engineered protein productionsystems. Such isolated molecules are those that are separated from theirnatural environment and include cDNA and genomic clones. Isolated DNAmolecules of the present invention are free of other genes with whichthey are ordinarily associated, but may include naturally occurring 5′and 3′ untranslated regions such as promoters and terminators. Theidentification of associated regions will be evident to one of ordinaryskill in the art (see for example, Dynan and Tijan, Nature 316:774-78,1985).

An “isolated” polypeptide or protein is a polypeptide or protein that isfound in a condition other than its native environment, such as apartfrom blood and animal tissue. In a preferred form, the isolatedpolypeptide is substantially free of other polypeptides, particularlyother polypeptides of animal origin. It is preferred to provide thepolypeptides in a highly purified form, i.e. greater than 95% pure, morepreferably greater than 99% pure. When used in this context, the term“isolated” does not exclude the presence of the same polypeptide inalternative physical forms, such as dimers, multimers, or alternativelyglycosylated or derivatized forms.

The term “operably linked”, when referring to DNA segments, indicatesthat the segments are arranged so that they function in concert fortheir intended purposes, e.g., transcription initiates in the promoterand proceeds through the coding segment to the terminator.

The term “ortholog” denotes a polypeptide or protein obtained from onespecies that is the functional counterpart of a polypeptide or proteinfrom a different species. Sequence differences among orthologs are theresult of speciation.

“Paralogs” are distinct but structurally related proteins made by anorganism. Paralogs are believed to arise through gene duplication. Forexample, α-globin, β-globin, and myoglobin are paralogs of each other.

A “polynucleotide” is a single- or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules. Sizes of polynucleotides are expressedas base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases(“kb”). Where the context allows, the latter two terms may describepolynucleotides that are single-stranded or double-stranded. When theterm is applied to double-stranded molecules it is used to denoteoverall length and will be understood to be equivalent to the term “basepairs”. It will be recognized by those skilled in the art that the twostrands of a double-stranded polynucleotide may differ slightly inlength and that the ends thereof may be staggered as a result ofenzymatic cleavage; thus all nucleotides within a double-strandedpolynucleotide molecule may not be paired.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides”.

“Probes and/or primers” as used herein can be RNA or DNA. DNA can beeither cDNA or genomic DNA. Polynucleotide probes and primers are singleor double-stranded DNA or RNA, generally synthetic oligonucleotides, butmay be generated from cloned cDNA or genomic sequences or itscomplements. Analytical probes will generally be at least 20 nucleotidesin length, although somewhat shorter probes (14-17 nucleotides) can beused. PCR primers are at least 5 nucleotides in length, preferably 15 ormore nt, more preferably 20-30 nt. Short polynucleotides can be usedwhen a small region of the gene is targeted for analysis. For grossanalysis of genes, a polynucleotide probe may comprise an entire exon ormore. Probes can be labeled to provide a detectable signal, such as withan enzyme, biotin, a radionuclide, fluorophore, chemiluminescer,paramagnetic particle and the like, which are commercially availablefrom many sources, such as Molecular Probes, Inc., Eugene, Oreg., andAmersham Corp., Arlington Heights, Ill., using techniques that are wellknown in the art.

The term “promoter” is used herein for its art-recognized meaning todenote a portion of a gene containing DNA sequences that provide for thebinding of RNA polymerase and initiation of transcription. Promotersequences are commonly, but not always, found in the 5′ non-codingregions of genes.

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

The term “receptor” is used herein to denote a cell-associated protein,or a polypeptide subunit of such a protein, that binds to a bioactivemolecule (the “ligand”) and mediates the effect of the ligand on thecell. Binding of ligand to receptor results in a conformational changein the receptor (and, in some cases, receptor multimerization, i.e.,association of identical or different receptor subunits) that causesinteractions between the effector domain(s) and other molecule(s) in thecell. These interactions in turn lead to alterations in the metabolismof the cell. Metabolic events that are linked to receptor-ligandinteractions include gene transcription, phosphorylation,dephosphorylation, cell proliferation, increases in cyclic AMPproduction, mobilization of cellular calcium, mobilization of membranelipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis ofphospholipids. Cytokine receptor subunits are characterized by amulti-domain structure comprising an extracellular domain, atransmembrane domain that anchors the polypeptide in the cell membrane,and an intracellular domain. The extracellular domain is typically aligand-binding domain, and the intracellular domain is typically aneffector domain involved in signal transduction, although ligand-bindingand effector functions may reside on separate subunits of a multimericreceptor. The ligand-binding domain may itself be a multi-domainstructure. Multimeric receptors include homodimers (e.g., PDGF receptorαα and ββ isoforms, erythropoietin receptor, MPL, and G-CSF receptor),heterodimers whose subunits each have ligand-binding and effectordomains (e.g., PDGF receptor αβ isoform), and multimers having componentsubunits with disparate functions (e.g., IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, and GM-CSF receptors). Some receptor subunits are common to aplurality of receptors. For example, the AIC2B subunit, which cannotbind ligand on its own but includes an intracellular signal transductiondomain, is a component of IL-3 and GM-CSF receptors. Many cytokinereceptors can be placed into one of four related families on the basisof the structure and function. Hematopoietic receptors, for example, arecharacterized by the presence of a domain containing conserved cysteineresidues and the WSXWS motif (SEQ ID NO:5). Cytokine receptor structurehas been reviewed by Urdal, Ann. Reports Med. Chem. 26:221-228, 1991 andCosman, Cytokine 5:95-106, 1993. Under selective pressure for organismsto acquire new biological functions, new receptor family members likelyarise from duplication of existing receptor genes leading to theexistence of multi-gene families. Family members thus contain vestigesof the ancestral gene, and these characteristic features can beexploited in the isolation and identification of additional familymembers. Thus, the cytokine receptor superfamily is subdivided intoseveral families, for example, the immunoglobulin family (includingCSF-1, MGF, IL-1, and PDGF receptors); the hematopoietin family(including IL-2 receptor β-subunit, GM-CSF receptor α-subunit, GM-CSFreceptor β-subunit; and G-CSF, EPO, IL-3, IL-4, IL-5, IL-6, IL-7, andIL-9 receptors); TNF receptor family (including TNF (p80) TNF (p60)receptors, CD27, CD30, CD40, Fas, and NGF receptor).

The term “receptor polypeptide” is used to denote complete receptorpolypeptide chains and portions thereof, including isolated functionaldomains (e.g., ligand-binding domains). The terms “ligand-bindingdomain(s)” and “cytokine-binding domain(s)” can be used interchangeably.

A “secretory signal sequence” is a DNA sequence that encodes apolypeptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger peptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

A “soluble receptor” is a receptor polypeptide that is not bound to acell membrane. Soluble receptors are most commonly ligand-bindingreceptor polypeptides that lack transmembrane and cytoplasmic domains.Soluble receptors can comprise additional amino acid residues, such asaffinity tags that provide for purification of the polypeptide orprovide sites for attachment of the polypeptide to a substrate, orimmunoglobulin constant region sequences. Many cell-surface receptorshave naturally occurring, soluble counterparts that are produced byproteolysis. Soluble receptor polypeptides are said to be substantiallyfree of transmembrane and intracellular polypeptide segments when theylack sufficient portions of these segments to provide membrane anchoringor signal transduction, respectively.

The term “splice variant” is used herein to denote alternative forms ofRNA transcribed from a gene. Splice variation arises naturally throughuse of alternative splicing sites within a transcribed RNA molecule, orless commonly between separately transcribed RNA molecules, and mayresult in several mRNAs transcribed from the same gene. Splice variantsmay encode polypeptides having altered amino acid sequence. The termsplice variant is also used herein to denote a protein encoded by asplice variant of an mRNA transcribed from a gene.

Molecular weights and lengths of polymers determined by impreciseanalytical methods (e.g., gel electrophoresis) will be understood to beapproximate values. When such a value is expressed as “about” X or“approximately” X, the stated value of X will be understood to beaccurate to ±10%.

All references cited herein are incorporated by reference in theirentirety.

Cytokine receptor subunits are characterized by a multi-domain structurecomprising a ligand-binding domain and an effector domain that istypically involved in signal transduction. Multimeric cytokine receptorsinclude homodimers (e.g., PDGF receptor αα and ββ isoforms,erythropoietin receptor, MPL (thrombopoietin receptor), and G-CSFreceptor); heterodimers whose subunits each have ligand-binding andeffector domains (e.g., PDGF receptor αβ isoform); and multimers havingcomponent subunits with disparate functions (e.g., IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, and GM-CSF receptors). Some receptor subunits arecommon to a plurality of receptors. For example, the AIC2B subunit,which cannot bind ligand on its own but includes an intracellular signaltransduction domain, is a component of IL-3 and GM-CSF receptors. Manycytokine receptors can be placed into one of four related families onthe basis of their structures and functions. Class I hematopoieticreceptors, for example, are characterized by the presence of a domaincontaining conserved cysteine residues and the WSXWS motif (SEQ IDNO:5). Additional domains, including protein kinase domains; fibronectintype III domains; and immunoglobulin domains, which are characterized bydisulfide-bonded loops, are present in certain hematopoietic receptors.Cytokine receptor structure has been reviewed by Urdal, Ann. ReportsMed. Chem. 26:221-228, 1991 and Cosman, Cytokine 5:95-106, 1993. It isgenerally believed that under selective pressure for organisms toacquire new biological functions, new receptor family members arose fromduplication of existing receptor genes leading to the existence ofmulti-gene families. Family members thus contain vestiges of theancestral gene, and these characteristic features can be exploited inthe isolation and identification of additional family members.

Cell-surface cytokine receptors are further characterized by thepresence of additional domains. These receptors are anchored in the cellmembrane by a transmembrane domain characterized by a sequence ofhydrophobic amino acid residues (typically about 21-25 residues), whichis commonly flanked by positively charged residues (Lys or Arg). On theopposite end of the protein from the extracellular domain and separatedfrom it by the transmembrane domain is an intracellular domain.

The Zcytor19 receptor of the present invention is a class II cytokinereceptor. These receptors usually bind to four-helix-bundle cytokines.Interleukin-10 and the interferons have receptors in this class (e.g.,interferon-gamma, alpha and beta chains and the interferon-alpha/betareceptor alpha and beta chains). Class II cytokine receptors arecharacterized by the presence of one or more cytokine receptor modules(CRM) in their extracellular domains. Other class II cytokine receptorsinclude zcytor11 (commonly owned U.S. Pat. No. 5,965,704), CRF2-4(Genbank Accession No. Z17227), IL-10R (Genbank Accession No.s U00672and NM_(—)001558), DIRS1, zcytor7 (commonly owned U.S. Pat. No.5,945,511), zcytor16, tissue factor, and the like. The CRMs of class IIcytokine receptors are somewhat different than the better-known CRMs ofclass I cytokine receptors. While the class II CRMs contain two type-IIIfibronectin-like domains, they differ in organization.

Zcytor19, like all known class II receptors except interferon-alpha/betareceptor alpha chain, has only a single class II CRM in itsextracellular domain. Zcytor19 is a receptor for a helical cytokine ofthe interferon/IL-10 class. As was stated above, Zcytor19 is similar toother Class II cytokine receptors such as zcytor11 and zcytor16. Due toits ability to bind IL28A, IL28B, and IL29 ligands, the Zcyto19 receptor(ZcytoR19) has been designated IL29RA.

Analysis of a human cDNA clone encoding Zcytor19 (SEQ ID NO:18) revealedan open reading frame encoding 520 amino acids (SEQ ID NO:19) comprisinga secretory signal sequence (residues 1 (Met) to 20 (Gly) of SEQ IDNO:19) and a mature zcytor19 cytokine receptor polypeptide (residues 21(Arg) to 520 (Arg) of SEQ ID NO:19) an extracellular ligand-bindingdomain of approximately 206 amino acid residues (residues 21 (Arg) to226 (Asn) of SEQ ID NO:19), a transmembrane domain of approximately 23amino acid residues (residues 227 (Trp) to 249 (Trp) of SEQ ID NO:19),and an intracellular domain of approximately 271 amino acid residues(residues 250 (Lys) to 520 (Arg) of SEQ ID NO:19). Within theextracellular ligand-binding domain, there are two fibronectin type IIIdomains and a linker region. The first fibronectin type III domaincomprises residues 21 (Arg) to 119 (Tyr) of SEQ ID NO:19, the linkercomprises residues 120 (Leu) to 124 (Glu) of SEQ ID NO:19, and thesecond fibronectin type III domain comprises residues 125 (Pro) to 223(Pro) of SEQ ID NO:19. Thus, a polypeptide comprising amino acids 21(Arg) to 223 (Pro) of SEQ ID NO:19 (SEQ ID NO:4) is considered a ligandbinding fragment. In addition as typically conserved in class IIreceptors, there are conserved Tryptophan residues comprising residues43 (Trp) and 68 (Trp) as shown in SEQ ID NO:19, and conserved Cysteineresidues at positions 74, 82, 195, 217 of SEQ ID NO:19.

In addition, analysis of a human cDNA clone encoding Zcytor19 (SEQ IDNO:1) revealed an open reading frame encoding 491 amino acids (SEQ IDNO:2) comprising a secretory signal sequence (residues 1 (Met) to 20(Gly) of SEQ ID NO:2) and a mature zcytor19 cytokine receptor polyptide(residues 21 (Arg) to 491 (Arg) of SEQ ID NO:2) an extracellularligand-binding domain of approximately 206 amino acid residues (residues21 (Arg) to 226 (Asn) of SEQ ID NO:2), a transmembrane domain ofapproximately 23 amino acid residues (residues 227 (Trp) to 249 (Trp) ofSEQ ID NO:2), and an intracellular domain of approximately 242 aminoacid residues (residues 250 (Lys) to 491 (Arg) of SEQ ID NO:2). Withinthe extracellular ligand-binding domain, there are two fibronectin typeIII domains and a linker region. The first fibronectin type III domaincomprises residues 21 (Arg) to 119 (Tyr) of SEQ ID NO:2, the linkercomprises residues 120 (Leu) to 124 (Glu) of SEQ ID NO:2, and the secondfibronectin type III domain is short, and comprises residues 125 (Pro)to 223 (Pro) of SEQ ID NO:2. Thus, a polypeptide comprising amino acids21 (Arg) to 223 (Pro) of SEQ ID NO:2 (SEQ ID NO:4) is considered aligand binding fragment. In addition as typically conserved in class IIreceptors, there are conserved. Tryptophan residues comprising residues43 (Trp) and 68 (Trp) as shown in SEQ ID NO:2, and conserved Cysteineresidues at positions 74, 82, 195, 217 of SEQ ID NO:2.

A truncated soluble form of the zcytor19 receptor mRNA appears to benaturally expressed. Analysis of a human cDNA clone encoding thetruncated soluble Zcytor19 (SEQ ID NO:20) revealed an open reading frameencoding 211 amino acids (SEQ ID NO:21) comprising a secretory signalsequence (residues 1 (Met) to 20 (Gly) of SEQ ID NO:21) and a maturetruncated soluble zcytor19 receptor polyptide (residues 21 (Arg) to 211(Ser) of SEQ ID NO:21) a truncated extracellular ligand-binding domainof approximately 143 amino acid residues (residues 21 (Arg) to 163 (Trp)of SEQ ID NO:21), no transmembrane domain, but an additional domain ofapproximately 48 amino acid residues (residues 164 (Lys) to 211 (Ser) ofSEQ ID NO:21). Within the truncated extracellular ligand-binding domain,there are two fibronectin type III domains and a linker region. Thefirst fibronectin type III domain comprises residues 21 (Arg) to 119(Tyr) of SEQ ID NO:21, the linker comprises residues 120 (Leu) to 124(Glu) of SEQ ID NO:21, and the second fibronectin type III domaincomprises residues 125 (Pro) to 163 (Trp) of SEQ ID NO:21. Thus, apolypeptide comprising amino acids 21 (Arg) to 163 (Tip) of SEQ ID NO:21is considered a ligand binding fragment. In addition as typicallyconserved in class II receptors, there are conserved Tryptophan residuescomprising residues 43 (Trp) and 68 (Trp) as shown in SEQ ID NO:21, andconserved Cysteine residues in this truncated soluble form of thezcytor19 receptor are at positions 74, and 82 of SEQ ID NO:21.

Moreover, the zcytor19 polypeptide of the present invention can benaturally expressed wherein the extracellular ligand binding domaincomprises an additional 5-15 amino acid residues at the N-terminus ofthe mature polypeptide, or extracellular cytokine binding domain orcytokine binding fragment, as described above.

Those skilled in the art will recognize that these domain boundaries areapproximate and are based on alignments with known proteins andpredictions of protein folding. Deletion of residues from the ends ofthe domains is possible. Moreover the regions, domains and motifsdescribed above in reference to SEQ ID NO:2 are also as shown in SEQ IDNO:1; domains and motifs described above in reference to SEQ ID NO:19are also as shown in SEQ ID NO:18; and domains and motifs describedabove in reference to SEQ ID NO:21 are also as shown in SEQ ID NO:20.

The presence of transmembrane regions, and conserved and low variancemotifs generally correlates with or defines important structural regionsin proteins. Regions of low variance (e.g., hydrophobic clusters) aregenerally present in regions of structural importance (Sheppard, P. etal., Gene 150:163-167, 1994). Such regions of low variance often containrare or infrequent amino acids, such as Tryptophan. The regions flankingand between such conserved and low variance motifs may be more variable,but are often functionally significant because they may relate to ordefine important structures and activities such as binding domains,biological and enzymatic activity, signal transduction, cell-cellinteraction, tissue localization domains and the like.

Analysis of the zcytor19 sequence has revealed that it is a member ofthe same receptor subfamily as the class II cytokine receptors, forexample, interferon-gamma, alpha and beta chains and theinterferon-alpha/beta receptor alpha and beta chains, zcytor11 (commonlyowned U.S. Pat. No. 5,965,704), CRF2-4 (Genbank Accession No. Z17227),DIRS1, zcytor7 (commonly owned U.S. Pat. No. 5,945,511) receptors.Several members of the subfamily (e.g., receptors that bind interferon,IL-10, IL-19, and IL-TIF) combine with a second subunit (termed aβ-subunit) to bind ligand and transduce a signal. Specific β-subunitsassociate with a plurality of specific cytokine receptor subunits.Zcytor19 has been shown to form a heterodimer with CRF2-4.

CRF2-4 has also been shown to be a binding partner with zcytor11(IL-22R) to bind the IL-10, and the binding partner for zcytor11 to bindcytokine IL-TIF (See, WIPO publication WO 00/24758; Dumontier et al., J.Immunol. 164:1814-1819, 2000; Spencer, S D et al., J. Exp. Med.187:571-578, 1998; Gibbs, V C and Pennica Gene 186:97-101, 1997 (CRF2-4cDNA); Xie, M H et al., J. Biol. Chem. 275: 31335-31339, 2000; andKotenko, S V et al., J. Biol. Chem. manuscript in press M007837200;Dumoutier, L. et al., Proc. Nat'l. Acad. Sci. 97:10144-10149, 2000; LiuY et al, J Immunol. 152; 1821-1829, 1994 (IL-10R cDNA). Receptors inthis subfamily may also associate to form heterodimers that transduce asignal. As such, class II receptor complexes can be heterodimeric, ormultimeric. Thus, monomeric, homodimeric, heterodimeric and multimericreceptors comprising a zcytor19 subunit are encompassed by the presentinvention.

Using the methods discussed herein, one of ordinary skill in the art canidentify and/or prepare a variety of polypeptide fragments or variantsof SEQ ID NO:2 or SEQ ID NO:19 that retain the signal transduction orligand binding activity. For example, one can make a zcytor19 “solublereceptor” by preparing a variety of polypeptides that are substantiallyhomologous to the extracellular cytokine-binding domain (residues 21(Arg) to 226 (Asn) of SEQ ID NO:2 or SEQ ID NO:19), a cytokine-bindingfragment (e.g., residues 21 (Arg) to 223 (Pro) of SEQ ID NO:2 or SEQ IDNO:19; SEQ ID NO:4) or allelic variants or species orthologs thereof)and retain ligand-binding activity of the wild-type zcytor19 protein.Moreover, variant zcytor19 soluble receptors can be isolated. Suchpolypeptides may include additional amino acids from, for example, partor all of the transmembrane and intracellular domains. Such polypeptidesmay also include additional polypeptide segments as generally disclosedherein such as labels, affinity tags, and the like.

The receptors of the present invention have been shown to form complexeswith a genus of polynucleotide and polypeptide molecules that havefunctional and structural similarity to the interferons. In this newfamily, which includes molecules designated zcyto20 (SEQ ID NOS: 51 and52), zcyto21 (SEQ ID NOS: 54 and 55), zcyto22 (SEQ ID NOS: 56 and 57),zcyto24 (SEQ ID NOS: 59 and 60), zcyto25 (SEQ ID NOS: 61 and 62),zcyto20, 21, and 22 are human sequences and zcyto24 and 25 are mousesequences. Furthermore, certain biological activities have been shown tobe exhibited by each molecule in the family. These activities include,for example, antiviral activities and increasing circulating myeloidcell levels. While not wanting to be bound by theory, these moleculesappear to all signal through zcytor19 receptor via the same pathway.

Homology within the ligand family at the nucleotide and amino acidlevels is shown in Table 1, ranging from approximately 72% to 98% at thenucleotide level, and 51% to 97% at the amino acid level.

TABLE 1 nucleotide sequence identity zcyto20 zcyto22 zcyto21 zcyto24zcyto25 rat protein zcyto20 100 98.2 72.9 74.0 72.1 73.4 se- zcyto2296.0 100 73.0 73.9 71.9 72.9 quence zcyto21 66.5 67.5 100 64.9 62.9 64.6identity zcyto24 62.7 63.7 51.7 100 97.2 90.3 zcyto25 59.8 60.8 48.893.6 100 88.4

Table 2 is an illustration of the sequence identity between zcyto20,zcyto21, zcyto22, IFNα, IFNβ, IFNγ, and IL10 at the amino acid level.

TABLE 2 amino acid sequence identity Zcyto20 Zcyto22 Zcyto21 IFN□ IFN□IFN□ IL10 Zcyto20 100 Zcyto21 81 100 Zcyto22 96 74 100 IFN□ 17 16 17 100IFN□ 14 13 14 31 100 IFN□ 4 4 4 7 5 100 IL10 13 12 14 7 5 8 100

Zcyto20 gene encodes a polypeptide of 205 amino acids, as shown in SEQID NO:52. The signal sequence for Zcyto20 can be predicted as comprisingamino acid residue 1 (Met) through amino acid residue 21 (Ala) of SEQ IDNO: 52. The mature peptide for Zcyto20 begins at amino acid residue 22(Val).

Zcyto21 gene encodes a polypeptide of 200 amino acids, as shown in SEQID NO:55. The signal sequence for Zcyto21 can be predicted as comprisingamino acid residue 1 (Met) through amino acid residue 19 (Ala) of SEQ IDNO: 55. The mature peptide for Zcyto21 begins at amino acid residue 20(Gly). Zcyto21 has been described in PCT application WO 02/02627.

Zcyto22 gene encodes a polypeptide of 205 amino acids, as shown in SEQID NO:57. The signal sequence for Zcyto22 can be predicted as comprisingamino acid residue 1 (Met) through amino acid residue 21 (Ala) of SEQ IDNO: 57. The mature peptide for Zcyto22 begins at amino acid residue 22(Val).

Zcyto24 gene encodes a polypeptide of 202 amino acids, as shown in SEQID NO:60. Zcyto24 secretory signal sequence comprises amino acid residue1 (Met) through amino acid residue 28 (Ala) of SEQ ID NO:60. Analternative site for cleavage of the secretory signal sequence can befound at amino acid residue 24 (Thr). The mature polypeptide comprisesamino acid residue 29 (Asp) to amino acid residue 202 (Val).

Zcyto25 gene encodes a polypeptide of 202 amino acids, as shown in SEQID NO:62. Zcyto25 secretory signal sequence comprises amino acid residue1 (Met) through amino acid residue 28 (Ala) of SEQ ID NO:62. Analternative site for cleavage of the secretory signal sequence can befound at amino acid residue 24 (Thr). The mature polypeptide comprisesamino acid residue 29 (Asp) to amino acid residue 202 (Val).

Evidence that CRF2-4 (SEQ ID NOS: 63 and 64) is the putative signalingpartner for zcytor19 provides further support that the receptor plays animportant role in the immunomodulatory system, affecting physiologiessuch as the innate immune system and the inflammatory response system.

Localizing the expression of a receptor for a ligand/receptor pair mayhave significance for identifying the target cell or tissue at which theligand acts. This is particularly useful when the receptor/ligandcomplex involves a heterodimeric receptor in which one of the subunitsis expressed widely and another of the subunits is expressed in alimited manner, either spatially or temporally restricted. Using in situhybridization expression of zcytor19 has been identified in a skincarcinoma sample, where the cancerous granular epithelium was stronglypositive, while no positive signal is observed in normal skin. Othertissues identified as expressing zcytor19 included fetal liver, wheresignal was observed in a mixed population of mononuclear cells insinusoid spaces; in lung expression was observed in type II alveolarepithelium; and in macrophage-like mononuclear cells in the interstitialtissue. Northern analysis of zcytor19 identified expression of a ˜4.5 kbtranscript which was in greatest in heart, skeletal muscle, pancreas,and prostate tissue, in addition to in the Burkitt's lymphoma (RAJI)cell line and SW-480 colorectal carcinoma cell line.

The regions of conserved amino acid residues in zcytor19, describedabove, can be used as tools to identify new family members. Forinstance, reverse transcription-polymerase chain reaction (RT-PCR) canbe used to amplify sequences encoding the conserved regions from RNAobtained from a variety of tissue sources or cell lines. In particular,highly degenerate primers designed from the zcytor19 sequences areuseful for this purpose. Designing and using such degenerate primers maybe readily performed by one of skill in the art.

The present invention provides polynucleotide molecules, including DNAand RNA molecules that encode the zcytor19 polypeptides disclosedherein. Those skilled in the art will recognize that, in view of thedegeneracy of the genetic code, considerable sequence variation ispossible among these polynucleotide molecules. SEQ ID NO:3 is adegenerate DNA sequence that encompass all DNAs that encode the zcytor19polypeptide of SEQ ID NO:2; SEQ ID NO:28 is a degenerate DNA sequencethat encompass all DNAs that encode the zcytor19 polypeptide of SEQ IDNO:19; and SEQ ID NO:29 is a degenerate DNA sequence that encompass allDNAs that encode the zcytor19 polypeptide of SEQ ID NO:21. Those skilledin the art will recognize that the degenerate sequences of SEQ ID NO:3,SEQ ID NO:28, and SEQ ID NO:29 also provide all RNA sequences encodingSEQ ID NO:2, SEQ ID NO:19, and SEQ ID NO:21 by substituting U for T.Thus, zcytor19 polypeptide-encoding polynucleotides comprisingnucleotide 1 to nucleotide 1473 of SEQ ID NO:3, 1 to nucleotide 1560 ofSEQ ID NO:28, 1 to nucleotide 633 of SEQ ID NO:29, and their RNAequivalents are contemplated by the present invention. Moreover,subfragments of these degenerate sequences such as the mature forms ofthe polypeptides, extracellular, cytokine binding domains, intracellulardomains, and the like, as described herein are included in the presentinvention. One of skill in the art upon reference to SEQ ID NO:2, SEQ IDNO:19 and SEQ ID NO:21 and the subfragments thereof described hereincould readily determine the respective nucleotides in SEQ ID NO:3, SEQID NO:28 or SEQ ID NO:29, that encode those subfragments. Table 3 setsforth the one-letter codes used within SEQ ID NO:3, to denote degeneratenucleotide positions. “Resolutions” are the nucleotides denoted by acode letter. “Complement” indicates the code for the complementarynucleotide(s). For example, the code Y denotes either C or T, and itscomplement R denotes A or G, A being complementary to T, and G beingcomplementary to C.

TABLE 3 Nucleotide Resolution Complement  Resolution A A T T C C G G G GC C T T A A R A|G Y C|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G S C|GW A|T W A|T H A|C|T D A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T HA|C|T N A|C|G|T N A|C|G|T

The degenerate codons used in SEQ ID NOs:3, 28, 29, 53, and 58encompassing all possible codons for a given amino acid, are set forthin Table 4.

TABLE 4 One  Amino Letter Degenerate Acid Code Codons Codon Cys CTGC TGT TGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT ACNPro P CCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly GGGA GGC GGG GGT GGN Asn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAGGAR Gln Q CAA CAG CAR His H CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGTMGN Lys K AAA AAG AAR Met M ATG ATG Ile I ATA ATC ATT ATH Leu LCTA CTC CTG CTT TTA TTG YTN Val V GTA GTC GTG GTT GTN Phe F TTC TTT TTYTyr Y TAC TAT TAY Trp W TGG TGG Ter . TAA TAG TGA TRR Asn|Asp B RAYGlu|Gln Z SAR Any X NNN

One of ordinary skill in the art will appreciate that some ambiguity isintroduced in determining a degenerate codon, representative of allpossible codons encoding each amino, acid. For example, the degeneratecodon for serine (WSN) can, in some circumstances, encode arginine(AGR), and the degenerate codon for arginine (MGN) can, in somecircumstances, encode serine (AGY). A similar relationship existsbetween codons encoding phenylalanine and leucine. Thus, somepolynucleotides encompassed by the degenerate sequence may encodevariant amino acid sequences, but one of ordinary skill in the art caneasily identify such variant sequences by reference to the amino acidsequence of SEQ ID NO:2, SEQ ID NO:19, and/or SEQ ID NO:21. Variantsequences can be readily tested for functionality as described herein.

One of ordinary skill in the art will also appreciate that differentspecies can exhibit “preferential codon usage.” In general, see,Grantham, et al., Nuc. Acids Res. 8:1893-912, 1980; Haas, et al. Curr.Biol. 6:315-24, 1996; Wain-Hobson, et al., Gene 13:355-64, 1981;Grosjean and Fiers, Gene 18:199-209, 1982; Hohn, Nuc. Acids Res.14:3075-87, 1986; Ikemura, J. Mol. Biol. 158:573-97, 1982. As usedherein, the term “preferential codon usage” or “preferential codons” isa term of art referring to protein translation codons that are mostfrequently used in cells of a certain species, thus favoring one or afew representatives of the possible codons encoding each amino acid (SeeTable 2). For example, the amino acid Threonine (Thr) may be encoded byACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonlyused codon; in other species, for example, insect cells, yeast, virusesor bacteria, different Thr codons may be preferential. Preferentialcodons for a particular species can be introduced into thepolynucleotides of the present invention by a variety of methods knownin the art. Introduction of preferential codon sequences intorecombinant DNA can, for example, enhance production of the protein bymaking protein translation more efficient within a particular cell typeor species. Therefore, the degenerate codon sequence disclosed in SEQ IDNO:3 serves as templates for optimizing expression of zcytor19polynucleotides in various cell types and species commonly used in theart and disclosed herein. Sequences containing preferential codons canbe tested and optimized for expression in various species, and testedfor functionality as disclosed herein.

Within preferred embodiments of the invention the isolatedpolynucleotides will hybridize to similar sized regions of SEQ ID NO:1,SEQ ID NO:18, or SEQ ID NO:20, or a sequence complementary thereto,under stringent conditions. In general, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence at a defined ionic strength and pH. The T_(m)is the temperature (under defined ionic strength and pH) at which 50% ofthe target sequence hybridizes to a perfectly matched probe. Numerousequations for calculating T_(m) are known in the art, and are specificfor DNA, RNA and DNA-RNA hybrids and polynucleotide probe sequences ofvarying length (see, for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition (Cold Spring Harbor Press 1989);Ausubel et al., (eds.), Current Protocols in Molecular Biology (JohnWiley and Sons, Inc. 1987); Berger and Kimmel (eds.), Guide to MolecularCloning Techniques, (Academic Press, Inc. 1987); and Wetmur, Crit. Rev.Biochem. Mol. Biol. 26:227 (1990)). Sequence analysis software such asOLIGO 6.0 (LSR; Long Lake, Minn.) and Primer Premier 4.0 (PremierBiosoft International; Palo Alto, Calif.), as well as sites on theInternet, are available tools for analyzing a given sequence andcalculating T_(m) based on user defined criteria. Such programs can alsoanalyze a given sequence under defined conditions and identify suitableprobe sequences. Typically, hybridization of longer polynucleotidesequences (e.g., >50 base pairs) is performed at temperatures of about20-25° C. below the calculated T_(m). For smaller probes (e.g., <50 basepairs) hybridization is typically carried out at the T_(m) or 5-10° C.below. This allows for the maximum rate of hybridization for DNA-DNA andDNA-RNA hybrids. Higher degrees of stringency at lower temperatures canbe achieved with the addition of formamide which reduces the T_(m) ofthe hybrid about 1° C. for each 1% formamide in the buffer solution.Suitable stringent hybridization conditions are equivalent to about a 5h to overnight incubation at about 42° C. in a solution comprising:about 40-50% formamide, up to about 6×SSC, about 5×Denhardt's solution,zero up to about 10% dextran sulfate, and about 10-20 μg/ml denaturedcommercially-available carrier DNA. Generally, such stringent conditionsinclude temperatures of 20-70° C. and a hybridization buffer containingup to 6×SSC and 0-50% formamide; hybridization is then followed bywashing filters in up to about 2×SSC. For example, a suitable washstringency is equivalent to 0.1×SSC to 2×SSC, 0.1% SDS, at 55° C. to 65°C. Different degrees of stringency can be used during hybridization andwashing to achieve maximum specific binding to the target sequence.Typically, the washes following hybridization are performed atincreasing degrees of stringency to remove non-hybridized polynucleotideprobes from hybridized complexes. Stringent hybridization and washconditions depend on the length of the probe, reflected in the Tm,hybridization and wash solutions used, and are routinely determinedempirically by one of skill in the art.

As previously noted, the isolated polynucleotides of the presentinvention include DNA and RNA. Methods for preparing DNA and RNA arewell known in the art. In general, RNA is isolated from a tissue or cellthat produces large amounts of zcytor19 RNA. Such tissues and cells areidentified by Northern blotting (Thomas, Proc. Natl. Acad. Sci. USA77:5201, 1980), and include PBLs, spleen, thymus, bone marrow, and lymphtissues, human erythroleukemia cell lines, acute monocytic leukemia celllines, B-cell and T-cell leukemia tissue or cell lines, other lymphoidand hematopoietic cell lines, and the like. Total RNA can be preparedusing guanidinium isothiocyanate extraction followed by isolation bycentrifugation in a CsCl gradient (Chirgwin et al., Biochemistry18:52-94, 1979). Poly (A)⁺ RNA is prepared from total RNA using themethod of Aviv and Leder (Proc. Natl. Acad. Sci. USA 69:1408-12, 1972).Complementary DNA (cDNA) is prepared from poly(A)⁺ RNA using knownmethods. In the alternative, genomic DNA can be isolated.Polynucleotides encoding zcytor19 polypeptides are then identified andisolated by, for example, hybridization or polymerase chain reaction(PCR) (Mullis, U.S. Pat. No. 4,683,202).

A full-length clone encoding zcytor19 can be obtained by conventionalcloning procedures. Complementary DNA (cDNA) clones are preferred,although for some applications (e.g., expression in transgenic animals)it may be preferable to use a genomic clone, or to modify a cDNA cloneto include at least one genomic intron. Methods for preparing cDNA andgenomic clones are well known and within the level of ordinary skill inthe art, and include the use of the sequence disclosed herein, or partsthereof, for probing or priming a library. Expression libraries can beprobed with antibodies to zcytor19, receptor fragments, or otherspecific binding partners.

The polynucleotides of the present invention can also be synthesizedusing DNA synthesis machines. Currently the method of choice is thephosphoramidite method. If chemically synthesized double stranded DNA isrequired for an application such as the synthesis of a gene or a genefragment, then each complementary strand is made separately. Analternative way to prepare a full-length gene is to synthesize aspecified set of overlapping oligonucleotides (40 to 100 nucleotides).See Glick and Pasternak, Molecular Biotechnology, Principles &Applications of Recombinant DNA, (ASM Press, Washington, D.C. 1994);Itakura et al., Annu. Rev. Biochem. 53: 323-56, 1984 and Climie et al.,Proc. Natl. Acad. Sci. USA 87:633-7, 1990. Moreover, other sequences aregenerally added that contain signals for proper initiation andtermination of transcription and translation.

The present invention further provides counterpart polypeptides andpolynucleotides from other species (orthologs). These species include,but are not limited to mammalian, avian, amphibian, reptile, fish,insect and other vertebrate and invertebrate species. Of particularinterest are zcytor19 polypeptides from other mammalian species,including murine, porcine, ovine, bovine, canine, feline, equine, andother primate polypeptides. Orthologs of human zcytor19 can be clonedusing information and compositions provided by the present invention incombination with conventional cloning techniques.

Those skilled in the art will recognize that the sequence disclosed inSEQ ID NO:1, SEQ ID NO:18, or SEQ ID NO:20 represents one allele ofhuman zcytor19 and that allelic variation and alternative splicing areexpected to occur. Allelic variants of this sequence can be cloned byprobing cDNA or genomic libraries from different individuals accordingto standard procedures. Allelic variants of the DNA sequence shown inSEQ ID NO:1, SEQ ID NO:18 or SEQ ID NO:20 including those containingsilent mutations and those in which mutations result in amino acidsequence changes, are within the scope of the present invention, as areproteins which are allelic variants of SEQ ID NO:2, SEQ ID NO:19 or SEQID NO:21. cDNAs generated from alternatively spliced mRNAs, which retainthe properties of the zcytor19 polypeptide are included within the scopeof the present invention, as are polypeptides encoded by such cDNAs andmRNAs. Allelic variants and splice variants of these sequences can becloned by probing cDNA or genomic libraries from different individualsor tissues according to standard procedures known in the art.

The present invention also provides isolated zcytor19 polypeptides thatare substantially similar to the polypeptides of SEQ ID NO:2, SEQ IDNO:19 or SEQ ID NO:21 and their orthologs. The term “substantiallysimilar” is used herein to denote polypeptides having at least 70%, morepreferably at least 80%, sequence identity to the sequences shown in SEQID NO:2, SEQ ID NO:19 or SEQ ID NO:21 or their orthologs. Suchpolypeptides will more preferably be at least 90% identical, and mostpreferably 95% or more identical to SEQ ID NO:2, SEQ ID NO:19 or SEQ IDNO:21 or its orthologs.) Percent sequence identity is determined byconventional methods. See, for example, Altschul et al., Bull. Math.Bio. 48: 603-616, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci.USA 89:10915-10919, 1992. Briefly, two amino acid sequences are alignedto optimize the alignment scores using a gap opening penalty of 10, agap extension penalty of 1, and the “blosum 62” scoring matrix ofHenikoff and Henikoff (ibid.) as shown in Table 5 (amino acids areindicated by the standard one-letter codes). The percent identity isthen calculated as:

$\frac{{Total}\mspace{14mu} {number}\mspace{14mu} {of}\mspace{14mu} {identical}\mspace{14mu} {matches}}{\begin{bmatrix}{{length}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {longer}\mspace{14mu} {sequence}\mspace{14mu} {plus}\mspace{14mu} {the}} \\{{number}\mspace{14mu} {of}\mspace{14mu} {gaps}\mspace{14mu} {introduced}\mspace{14mu} {into}\mspace{14mu} {the}\mspace{14mu} {longer}} \\{{sequence}\mspace{14mu} {in}\mspace{14mu} {order}\mspace{14mu} {to}\mspace{14mu} {align}\mspace{14mu} {the}\mspace{14mu} {two}\mspace{14mu} {sequences}}\end{bmatrix}} \times 100$

TABLE 5 A R N D C Q E G H I L K M F P S T W Y V A 4 R −1 5 N −2 0 6 D −2−2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 2 5 G 0 −2 0 −1 −3−2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 L −1 −2−3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 M −1 −1 −2 −3−1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 P −1 −2−2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1−2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W −3 −3 −4 −4−2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1 −2 −3 2 −1−1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 −2 0−3 −1 4

Sequence identity of polynucleotide molecules is determined by similarmethods using a ratio as disclosed above.

Those skilled in the art appreciate that there are many establishedalgorithms available to align two amino acid sequences. The “FASTA”similarity search algorithm of Pearson and Lipman is a suitable proteinalignment method for examining the level of identity shared by an aminoacid sequence disclosed herein and the amino acid sequence of a putativevariant zcytor19. The FASTA algorithm is described by Pearson andLipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth.Enzymol. 183:63 (1990).

Briefly, FASTA first characterizes sequence similarity by identifyingregions shared by the query sequence (e.g., SEQ ID NO:2, SEQ ID NO:19 orSEQ ID NO:21) and a test sequence that have either the highest densityof identities (if the ktup variable is 1) or pairs of identities (iflaity-2), without considering conservative amino acid substitutions,insertions, or deletions. The ten regions with the highest density ofidentities are then rescored by comparing the similarity of all pairedamino acids using an amino acid substitution matrix, and the ends of theregions are “trimmed” to include only those residues that contribute tothe highest score. If there are several regions with scores greater thanthe “cutoff” value (calculated by a predetermined formula based upon thelength of the sequence and the ktup value), then the trimmed initialregions are examined to determine whether the regions can be joined toform an approximate alignment with gaps. Finally, the highest scoringregions of the two amino acid sequences are aligned using a modificationof the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol.Biol. 48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), whichallows for amino acid insertions and deletions. Preferred parameters forFASTA analysis are: ktup=1, gap opening penalty=10, gap extensionpenalty=1, and substitution matrix=BLOSUM62, with other parameters setas default. These parameters can be introduced into a FASTA program bymodifying the scoring matrix file (“SMATRIX”), as explained in Appendix2 of Pearson, Meth. Enzymol. 183:63 (1990).

FASTA can also be used to determine the sequence identity of nucleicacid molecules using a ratio as disclosed above. For nucleotide sequencecomparisons, the ktup value can range between one to six, preferablyfrom three to six, most preferably three, with other FASTA programparameters set as default.

The BLOSUM62 table (Table 3) is an amino acid substitution matrixderived from about 2,000 local multiple alignments of protein sequencesegments, representing highly conserved regions of more than 500 groupsof related proteins (Henikoff and Henikoff, Proc. Nat'l Acad. Sci. USA89:10915 (1992)). Accordingly, the BLOSUM62 substitution frequencies canbe used to define conservative amino acid substitutions that may beintroduced into the amino acid sequences of the present invention.Although it is possible to design amino acid substitutions based solelyupon chemical properties (as discussed below), the language“conservative amino acid substitution” preferably refers to asubstitution represented by a BLOSUM62 value of greater than −1. Forexample, an amino acid substitution is conservative if the substitutionis characterized by a BLOSUM62 value of 0, 1, 2, or 3. According to thissystem, preferred conservative amino acid substitutions arecharacterized by a BLOSUM62 value of at least 1 (e.g., 1, 2 or 3), whilemore preferred conservative amino acid substitutions are characterizedby a BLOSUM62 value of at least 2 (e.g., 2 or 3).

Variant zcytor19 polypeptides or substantially homologous zcytor19polypeptides are characterized as having one or more amino acidsubstitutions, deletions or additions. These changes are preferably of aminor nature, that is conservative amino acid substitutions (see Table6) and other substitutions that do not significantly affect the foldingor activity of the polypeptide; small deletions, typically of one toabout 30 amino acids; and small amino- or carboxyl-terminal extensions,such as an amino-terminal methionine residue, a small linker peptide ofup to about 20-25 residues, or an affinity tag. The present inventionthus includes polypeptides that comprise a sequence that is at least80%, preferably at least 90%, and more preferably 95% or more identicalto the corresponding region of SEQ ID NO:2, SEQ ID NO:19 or SEQ IDNO:21, excluding the tags, extension, linker sequences and the like.Polypeptides comprising affinity tags can further comprise a proteolyticcleavage site between the zcytor19 polypeptide and the affinity tag.Suitable sites include thrombin cleavage sites and factor Xa cleavagesites.

TABLE 6 Conservative amino acid substitutions Basic: arginine lysinehistidine Acidic: glutamic acid aspartic acid Polar: glutamineasparagine Hydrophobic: leucine isoleucine valine Aromatic:phenylalanine tryptophan tyrosine Small: glycine alanine serinethreonine methionine

The present invention further provides a variety of other polypeptidefusions and related multimeric proteins comprising one or morepolypeptide fusions. For example, a zcytor19 polypeptide can be preparedas a fusion to a dimerizing protein as disclosed in U.S. Pat. Nos.5,155,027 and 5,567,584. Preferred dimerizing proteins in this regardinclude immunoglobulin constant region domains. Immunoglobulin-zcytor19polypeptide fusions can be expressed in genetically engineered cells toproduce a variety of multimeric zcytor19 analogs. Auxiliary domains canbe fused to zcytor19 polypeptides to target them to specific cells,tissues, or macromolecules (e.g., collagen). A zcytor19 polypeptide canbe fused to two or more moieties, such as an affinity tag forpurification and a targeting domain. Polypeptide fusions can alsocomprise one or more cleavage sites, particularly between domains. See,Tuan et al., Connective Tissue Research 34:1-9, 1996.

The proteins of the present invention can also comprise non-naturallyoccurring amino acid residues. Non-naturally occurring amino acidsinclude, without limitation, trans-3-methylproline, 2,4-methanoproline,cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine,alto-threonine, methylthreonine, hydroxyethylcysteine,hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid,thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline,3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine,3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.Several methods are known in the art for incorporating non-naturallyoccurring amino acid residues into proteins. For example, an in vitrosystem can be employed wherein nonsense mutations are suppressed usingchemically aminoacylated suppressor tRNAs. Methods for synthesizingamino acids and aminoacylating tRNA are known in the art. Transcriptionand translation of plasmids containing nonsense mutations is carried outin a cell-free system comprising an E. coli S30 extract and commerciallyavailable enzymes and other reagents. Proteins are purified bychromatography. See, for example, Robertson et al., J. Am. Chem. Soc.113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung etal., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci.USA 90:10145-9, 1993). In a second method, translation is carried out inXenopus oocytes by microinjection of mutated mRNA and chemicallyaminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem.271:19991-8, 1996). Within a third method, E. coli cells are cultured inthe absence of a natural amino acid that is to be replaced (e.g.,phenylalanine) and in the presence of the desired non-naturallyoccurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturallyoccurring amino acid is incorporated into the protein in place of itsnatural counterpart. See, Koide et al., Biochem. 33:7470-7476, 1994.Naturally occurring amino acid residues can be converted tonon-naturally occurring species by in vitro chemical modification.Chemical modification can be combined with site-directed mutagenesis tofurther expand the range of substitutions (Wynn and Richards, ProteinSci. 2:395-403, 1993).

A limited number of non-conservative amino acids, amino acids that arenot encoded by the genetic code, non-naturally occurring amino acids,and unnatural amino acids may be substituted for zcytor19 amino acidresidues.

Essential amino acids in the polypeptides of the present invention canbe identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunninghamand Wells, Science 244: 1081-5, 1989; Bass et al., Proc. Natl. Acad.Sci. USA 88:4498-502, 1991). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for biological activity (e.g.ligand binding and signal transduction) as disclosed below to identifyamino acid residues that are critical to the activity of the molecule.See also, Hilton et al., J. Biol. Chem. 271:4699-4708, 1996. Sites ofligand-receptor, protein-protein or other biological interaction canalso be determined by physical analysis of structure, as determined bysuch techniques as nuclear magnetic resonance, crystallography, electrondiffraction or photoaffinity labeling, in conjunction with mutation ofputative contact site amino acids. See, for example, de Vos et al.,Science 255:306-312, 1992; Smith et al., J. Mol. Biol. 224:899-904,1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identities ofessential amino acids can also be inferred from analysis of homologieswith related receptors.

Determination of amino acid residues that are within regions or domainsthat are critical to maintaining structural integrity can be determined.Within these regions one can determine specific residues that will bemore or less tolerant of change and maintain the overall tertiarystructure of the molecule. Methods for analyzing sequence structureinclude, but are not limited to, alignment of multiple sequences withhigh amino acid or nucleotide identity and computer analysis usingavailable software (e.g., the Insight II® viewer and homology modelingtools; MSI, San Diego, Calif.), secondary structure propensities, binarypatterns, complementary packing and buried polar interactions (Barton,Current Opin. Struct. Biol. 5:372-376, 1995 and Cordes et al., CurrentOpin. Struct. Biol. 6:3-10, 1996). In general, when designingmodifications to molecules or identifying specific fragmentsdetermination of structure will be accompanied by evaluating activity ofmodified molecules.

Amino acid sequence changes are made in zcytor19 polypeptides so as tominimize disruption of higher order structure essential to biologicalactivity. For example, when the zcytor19 polypeptide comprises one ormore structural domains, such as Fibronectin Type III domains, changesin amino acid residues will be made so as not to disrupt the domainstructure and geometry and other components of the molecule wherechanges in conformation ablate some critical function, for example,binding of the molecule to its binding partners. The effects of aminoacid sequence changes can be predicted by, for example, computermodeling as disclosed above or determined by analysis of crystalstructure (see, e.g., Lapthorn et al., Nat. Struct. Biol. 2:266-268,1995). Other techniques that are well known in the art compare foldingof a variant protein to a standard molecule (e.g., the native protein).For example, comparison of the cysteine pattern in a variant andstandard molecules can be made. Mass spectrometry and chemicalmodification using reduction and alkylation provide methods fordetermining cysteine residues which are associated with disulfide bondsor are free of such associations (Bean et al., Anal. Biochem.201:216-226, 1992; Gray, Protein Sci. 2:1732-1748, 1993; and Pattersonet al., Anal. Chem. 66:3727-3732, 1994). It is generally believed thatif a modified molecule does not have the same disulfide bonding patternas the standard molecule folding would be affected. Another well knownand accepted method for measuring folding is circular dichroism (CD).Measuring and comparing the CD spectra generated by a modified moleculeand standard molecule is routine (Johnson, Proteins 7:205-214, 1990).Crystallography is another well known method for analyzing folding andstructure. Nuclear magnetic resonance (NMR), digestive peptide mappingand epitope mapping are also known methods for analyzing folding andstructural similarities between proteins and polypeptides (Schaanan etal., Science 257:961-964, 1992).

A Hopp/Woods hydrophilicity profile of the zcytor19 protein sequence asshown in SEQ ID NO:2, SEQ ID NO:19 or SEQ ID NO:21 can be generated(Hopp et al., Proc. Natl. Acad. Sci. 78:3824-3828, 1981; Hopp, J. Immun.Meth. 88:1-18, 1986 and Triquier et al., Protein Engineering 11:153-169,1998). The profile is based on a sliding six-residue window. Buried G,S, and T residues and exposed H, Y, and W residues were ignored. Forexample, in zcytor19, hydrophilic regions include amino acid residues295 through 300 of SEQ ID NO:2; 451 through 456 of SEQ ID NO:2; 301through 306 of SEQ ID NO:2; 244 through 299 of SEQ ID NO:2; and 65through 70 of SEQ ID NO:2. Moreover, one of skill in the art wouldrecognize that zcytor19 hydrophilic regions including antigenicepitope-bearing polypeptides can be predicted by a Jameson-Wolf plot,e.g., using DNASTAR Protean program (DNASTAR, Inc., Madison, Wis.).

Those skilled in the art will recognize that hydrophilicity orhydrophobicity will be taken into account when designing modificationsin the amino acid sequence of a zcytor19 polypeptide, so as not todisrupt the overall structural and biological profile. Of particularinterest for replacement are hydrophobic residues selected from thegroup consisting of Val, Leu and Ile or the group consisting of Met,Gly, Ser, Ala, Tyr and Trp. For example, residues tolerant ofsubstitution could include such residues as shown in SEQ ID NO:2.However, Cysteine residues at positions 74, 82, 195, and 217 of SEQ IDNO:2 or SEQ ID NO:19, and corresponding Cys residues in SEQ ID NO:4 arerelatively intolerant of substitution. Moreover, Cysteine residues atpositions 74, 82, of SEQ ID NO:21 are relatively intolerant ofsubstitution.

The identities of essential amino acids can also be inferred fromanalysis of sequence similarity between class II cytokine receptorfamily members with zcytor19. Using methods such as “FASTA” analysisdescribed previously, regions of high similarity are identified within afamily of proteins and used to analyze amino acid sequence for conservedregions. An alternative approach to identifying a variant zcytor19polynucleotide on the basis of structure is to determine whether anucleic acid molecule encoding a potential variant zcytor19polynucleotide can hybridize to a nucleic acid molecule having thenucleotide sequence of SEQ ID NO:1, SEQ ID NO:18, or SEQ ID NO:20 asdiscussed above.

Other methods of identifying essential amino acids in the polypeptidesof the present invention are procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunninghamand Wells, Science 244:1081 (1989), Bass et al., Proc. Natl. Acad. Sci.USA 88:4498 (1991), Coombs and Corey, “Site-Directed Mutagenesis andProtein Engineering,” in Proteins: Analysis and Design, Angeletti (ed.),pages 259-311 (Academic Press, Inc. 1998)). In the latter technique,single alanine mutations are introduced at every residue in themolecule, and the resultant mutant molecules are tested for biologicalactivity as disclosed below to identify amino acid residues that arecritical to the activity of the molecule. Such mutagenesis and screeningmethods are routine in the art. See also, Hilton et al., J. Biol. Chem.271:4699 (1996).

The present invention also includes functional fragments of zcytor19polypeptides and nucleic acid molecules encoding such functionalfragments. A “functional” zcytor19 or fragment thereof defined herein ischaracterized by its proliferative or differentiating activity, by itsability to induce or inhibit specialized cell functions, or by itsability to bind specifically to an anti-zcytor19 antibody or zcytor19ligand (either soluble or immobilized). Moreover, functional fragmentsalso include the signal peptide, intracellular signaling domain, and thelike. As previously described herein, zcytor19 is characterized by aclass II cytokine receptor structure. Thus, the present inventionfurther provides fusion proteins encompassing: (a) polypeptide moleculescomprising an extracellular domain, cytokine-binding domain, orintracellular domain described herein; and (b) functional fragmentscomprising one or more of these domains. The other polypeptide portionof the fusion protein may be contributed by another class II cytokinereceptor, for example, interferon-gamma, alpha and beta chains and theinterferon-alpha/beta receptor alpha and beta chains, zcytor11 (commonlyowned U.S. Pat. No. 5,965,704), CRF2-4, DIRS1, zcytor7 (commonly ownedU.S. Pat. No. 5,945,511), and the like, or by a non-native and/or anunrelated secretory signal peptide that facilitates secretion of thefusion protein.

Routine deletion analyses of nucleic acid molecules can be performed toobtain functional fragments of a nucleic acid molecule that encodes azcytor19 polypeptide. As an illustration, DNA molecules having thenucleotide sequence of SEQ ID NO:1, SEQ ID NO:18, or SEQ ID NO:20 orfragments thereof, can be digested with Bal31 nuclease to obtain aseries of nested deletions. These DNA fragments are then inserted intoexpression vectors in proper reading frame, and the expressedpolypeptides are isolated and tested for zcytor19 activity, or for theability to bind anti-zcytor19 antibodies or zcytor19 ligand. Onealternative to exonuclease digestion is to use oligonucleotide-directedmutagenesis to introduce deletions or stop codons to specify productionof a desired zcytor19 fragment. Alternatively, particular fragments of azcytor19 polynucleotide can be synthesized using the polymerase chainreaction.

Standard methods for identifying functional domains are well-known tothose of skill in the art. For example, studies on the truncation ateither or both termini of interferons have been summarized byHorisberger and Di Marco, Pharmac. Ther. 66:507 (1995). Moreover,standard techniques for functional analysis of proteins are describedby, for example, Treuter et al., Molec. Gen. Genet. 240:113 (1993);Content et al., “Expression and preliminary deletion analysis of the 42kDa 2-5A synthetase induced by human interferon,” in BiologicalInterferon Systems, Proceedings of ISIR-TNO Meeting on InterferonSystems, Cantell (ed.), pages 65-72 (Nijhoff 1987); Herschman, “The EGFReceptor,” in Control of Animal Cell Proliferation 1, Boynton et al.,(eds.) pages 169-199 (Academic Press 1985); Coumailleau et al., J. Biol.Chem. 270:29270 (1995); Fukunaga et al., J. Biol. Chem. 270:25291(1995); Yamaguchi et al., Biochem. Pharmacol. 50:1295 (1995); and Meiselet al., Plant Molec. Biol. 30:1 (1996).

Multiple amino acid substitutions can be made and tested using knownmethods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53-57, 1988) or Bowie and Sauer(Proc. Natl. Acad. Sci. USA 86:2152-2156, 1989). Other methods that canbe used include phage display (e.g., Lowman et al., Biochem.30:10832-10837, 1991; Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPOPublication WO 92/062045) and region-directed mutagenesis (Derbyshire etal., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

Variants of the disclosed zcytor19 DNA and polypeptide sequences can begenerated through DNA shuffling as disclosed by Stemmer, Nature370:389-91, 1994, Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-51, 1994and WIPO Publication WO 97/20078.

Mutagenesis methods as disclosed herein can be combined withhigh-throughput, automated screening methods to detect activity ofcloned, mutagenized zcytor19 receptor polypeptides in host cells.Preferred assays in this regard include cell proliferation assays andbiosensor-based ligand-binding assays, which are described below.Mutagenized DNA molecules that encode active receptors or portionsthereof (e.g., ligand-binding fragments, signaling domains, and thelike) can be recovered from the host cells and rapidly sequenced usingmodern equipment. These methods allow the routine and rapiddetermination of the importance of individual amino acid residues in apolypeptide of interest.

In addition, the proteins of the present invention (or polypeptidefragments thereof) can be joined to other bioactive molecules,particularly cytokine receptors, to provide multi-functional molecules.For example, one or more domains from zcytor19 soluble receptor can bejoined to other cytokine soluble receptors to enhance their biologicalproperties or efficiency of production.

The present invention thus provides a series of novel, hybrid moleculesin which a segment comprising one or more of the domains of zcytor19 isfused to another polypeptide. Fusion is preferably done by splicing atthe DNA level to allow expression of chimeric molecules in recombinantproduction systems. The resultant molecules are then assayed for suchproperties as improved solubility, improved stability, prolongedclearance half-life, improved expression and secretion levels, andpharmacodynamics. Such hybrid molecules may further comprise additionalamino acid residues (e.g. a polypeptide linker) between the componentproteins or polypeptides.

For any zcytor19 polypeptide, including variants, soluble receptors, andfusion polypeptides or proteins, one of ordinary skill in the art canreadily generate a fully degenerate polynucleotide sequence encodingthat variant using the information set forth in Tables 1 and 2 above.

The zcytor19 polypeptides of the present invention, includingfull-length polypeptides, biologically active fragments, and fusionpolypeptides, can be produced in genetically engineered host cellsaccording to conventional techniques. Suitable host cells are those celltypes that can be transformed or transfected with exogenous DNA andgrown in culture, and include bacteria, fungal cells, and culturedhigher eukaryotic cells. Eukaryotic cells, particularly cultured cellsof multicellular organisms, are preferred. Techniques for manipulatingcloned DNA molecules and introducing exogenous DNA into a variety ofhost cells are disclosed by Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989, and Ausubel et al., eds., Current Protocolsin Molecular Biology, John Wiley and Sons, Inc., NY, 1987.

In general, a DNA sequence encoding a zcytor19 polypeptide is operablylinked to other genetic elements required for its expression, generallyincluding a transcription promoter and terminator, within an expressionvector. The vector will also commonly contain one or more selectablemarkers and one or more origins of replication, although those skilledin the art will recognize that within certain systems selectable markersmay be provided on separate vectors, and replication of the exogenousDNA may be provided by integration into the host cell genome. Selectionof promoters, terminators, selectable markers, vectors and otherelements is a matter of routine design within the level of ordinaryskill in the art. Many such elements are described in the literature andare available through commercial suppliers.

To direct a zcytor19 polypeptide into the secretory pathway of a hostcell, a secretory signal sequence (also known as a leader sequence,prepro sequence or pre sequence) is provided in the expression vector.The secretory signal sequence may be that of zcytor19, or may be derivedfrom another secreted protein (e.g., t-PA) or synthesized de novo. Thesecretory signal sequence is operably linked to the zcytor19 DNAsequence, i.e., the two sequences are joined in the correct readingframe and positioned to direct the newly synthesized polypeptide intothe secretory pathway of the host cell. Secretory signal sequences arecommonly positioned 5′ to the DNA sequence encoding the polypeptide ofinterest, although certain secretory signal sequences may be positionedelsewhere in the DNA sequence of interest (see, e.g., Welch et al., U.S.Pat. No. 5,037,743; Holland et al., U.S. Pat. No. 5,143,830).

Alternatively, the secretory signal sequence contained in thepolypeptides of the present invention is used to direct otherpolypeptides into the secretory pathway. The present invention providesfor such fusion polypeptides. A signal fusion polypeptide can be madewherein a secretory signal sequence derived from amino acid 1 (Met) toamino acid 20 (Gly) of SEQ ID NO:2 or SEQ ID NO:19 is operably linked toanother polypeptide using methods known in the art and disclosed herein.The secretory signal sequence contained in the fusion polypeptides ofthe present invention is preferably fused amino-terminally to anadditional peptide to direct the additional peptide into the secretorypathway.

Cultured mammalian cells are suitable hosts within the presentinvention. Methods for introducing exogenous DNA into mammalian hostcells include calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603,1981: Graham and Van der Eb, Virology 52:456, 1973), electroporation(Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextran mediatedtransfection (Ausubel et al., ibid.), and liposome-mediated transfection(Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80,1993, and viral vectors (Miller and Rosman, BioTechniques 7:980-90,1989; Wang and Finer, Nature Med. 2:714-716, 1996). The production ofrecombinant polypeptides in cultured mammalian cells is disclosed, forexample, by Levinson et al., U.S. Pat. No. 4,713,339; Hagen et al., U.S.Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No. 4,579,821; andRingold, U.S. Pat. No. 4,656,134. Suitable cultured mammalian cellsinclude the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK(ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and Chinese hamsterovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines. Additional suitablecell lines are known in the art and available from public depositoriessuch as the American Type Culture Collection (ATCC), Rockville, Md. Ingeneral, strong transcription promoters are preferred, such as promotersfrom SV-40 or cytomegalovirus. See, e.g., U.S. Pat. No. 4,956,288. Othersuitable promoters include those from metallothionein genes (U.S. Pat.Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.

Drug selection is generally used to select for cultured mammalian cellsinto which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants”. Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.” Apreferred selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems canalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.A preferred amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g. hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used. Alternative markers that introducean altered phenotype, such as green fluorescent protein, or cell surfaceproteins such as CD4, CD8, Class I MHC, placental alkaline phosphatasemay be used to sort transfected cells from untransfected cells by suchmeans as FACS sorting or magnetic bead separation technology.

Other higher eukaryotic cells can also be used as hosts, including plantcells, insect cells and avian cells. The use of Agrobacterium rhizogenesas a vector for expressing genes in plant cells has been reviewed bySinkar et al., J. Biosci. (Bangalore) 11:47-58, 1987. Transformation ofinsect cells and production of foreign polypeptides therein is disclosedby Guarino et al., U.S. Pat. No. 5,162,222 and WIPO publication WO94/06463. Insect cells can be infected with recombinant baculovirus,commonly derived from Autographa californica nuclear polyhedrosis virus(AcNPV). See, King, L. A. and Possee, R. D., The Baculovirus ExpressionSystem: A Laboratory Guide, London, Chapman & Hall; O'Reilly, D. R. etal., Baculovirus Expression Vectors: A Laboratory Manual, New York,Oxford University Press., 1994; and, Richardson, C. D., Ed., BaculovirusExpression Protocols. Methods in Molecular Biology, Totowa, N.J., HumanaPress, 1995. A second method of making recombinant zcytor19 baculovirusutilizes a transposon-based system described by Luckow (Luckow, V. A, etal., J Virol 67:4566-79, 1993). This system, which utilizes transfervectors, is sold in the Bac-to-Bac™ kit (Life Technologies, Rockville,Md.). This system utilizes a transfer vector, pFastBac1™ (LifeTechnologies) containing a Tn7 transposon to move the DNA encoding thezcytor19 polypeptide into a baculovirus genome maintained in E. coli asa large plasmid called a “bacmid.” See, Hill-Perkins, M. S, and Possee,R. D., J Gen Virol 71:971-6, 1990; Bonning, B. C. et al., J Gen Virol75:1551-6, 1994; and, Chazenbalk, G. D., and Rapoport, B., J Biol Chem270:1543-9, 1995. In addition, transfer vectors can include an in-framefusion with DNA encoding an epitope tag at the C- or N-terminus of theexpressed zcytor19 polypeptide, for example, a Glu-Glu epitope tag(Grussenmeyer, T. et al., Proc. Natl. Acad. Sci. 82:7952-4, 1985).

The recombinant virus is used to infect host cells, typically a cellline derived from the fall armyworm, Spodoptera frugiperda. See, ingeneral, Glick and Pasternak, Molecular Biotechnology: Principles andApplications of Recombinant DNA, ASM Press, Washington, D.C., 1994.Another suitable cell line is the High FiveO™ cell line (Invitrogen)derived from Trichoplusia ni (U.S. Pat. No. 5,300,435). Commerciallyavailable serum-free media are used to grow and maintain the cells.Suitable media are Sf900 II™ (Life Technologies) or ESF 921™ (ExpressionSystems) for the Sf9 cells; and Ex-cellO405™ (JRH Biosciences, Lenexa,Kans.) or Express FiveO™ (Life Technologies) for the T. ni cells.Procedures used are generally described in available laboratory manuals(King, L. A. and Possee, R. D., ibid.; O'Reilly, D. R. et al., ibid.;Richardson, C. D., ibid.). Subsequent purification of the zcytor19polypeptide from the supernatant can be achieved using methods describedherein.

Fungal cells, including yeast cells, can also be used within the presentinvention. Yeast species of particular interest in this regard includeSaccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica.Methods for transforming S. cerevisiae cells with exogenous DNA andproducing recombinant polypeptides therefrom are disclosed by, forexample, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat.No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat.No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075. Transformedcells are selected by phenotype determined by the selectable marker,commonly drug resistance or the ability to grow in the absence of aparticular nutrient (e.g., leucine). A preferred vector system for usein Saccharomyces cerevisiae is the POT1 vector system disclosed byKawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformedcells to be selected by growth in glucose-containing media. Suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman etal., U.S. Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092) andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446;5,063,154; 5,139,936 and 4,661,454. Transformation systems for otheryeasts, including Hansenula polymorpha, Schizosaccharomyces pombe,Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichiapastoris, Pichia methanolica, Pichia guillemmondii and Candida maltosaare known in the art. See, for example, Gleeson et al., J. Gen.Microbiol. 132:3459-3465, 1986 and Cregg, U.S. Pat. No. 4,882,279.Aspergillus cells may be utilized according to the methods of McKnightet al., U.S. Pat. No. 4,935,349. Methods for transforming Acremoniumchrysogenum are disclosed by Sumino et al., U.S. Pat. No. 5,162,228.Methods for transforming Neurospora are disclosed by Lambowitz, U.S.Pat. No. 4,486,533. The use of Pichia methanolica as host for theproduction of recombinant proteins is disclosed in WIPO Publications WO97/17450, WO 97/17451, WO 98/02536, and WO 98/02565.

Prokaryotic host cells, including strains of the bacteria Escherichiacoli, Bacillus and other genera are also useful host cells within thepresent invention. Techniques for transforming these hosts andexpressing foreign DNA sequences cloned therein are well known in theart (see, e.g., Sambrook et al., ibid.).

Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell. P. methanolicacells are cultured in a medium comprising adequate sources of carbon,nitrogen and trace nutrients at a temperature of about 25° C. to 35° C.Liquid cultures are provided with sufficient aeration by conventionalmeans, such as shaking of small flasks or sparging of fermentors. Apreferred culture medium for P. methanolica is YEPD (2% D-glucose, 2%Bacto™ Peptone (Difco Laboratories, Detroit, Mich.), 1% Bacto™ yeastextract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine).

Within one aspect of the present invention, a zcytor19 cytokine receptor(including transmembrane and intracellular domains) is produced by acultured cell, and the cell is used to screen for ligands for thereceptor, including the natural ligand, as well as agonists andantagonists of the natural ligand. To summarize this approach, a cDNA orgene encoding the receptor is combined with other genetic elementsrequired for its expression (e.g., a transcription promoter), and theresulting expression vector is inserted into a host cell. Cells thatexpress the DNA and produce functional receptor are selected and usedwithin a variety of screening systems.

Mammalian cells suitable for use in expressing the novel receptors ofthe present invention and transducing a receptor-mediated signal includecells that express a β-subunit, such as a class II cytokine receptorsubunit, for example, interferon-gamma, alpha and beta chains and theinterferon-alpha/beta receptor alpha and beta chains, zcytor11 (commonlyowned U.S. Pat. No. 5,965,704), CRF2-4, DIRS1, zcytor7 (commonly ownedU.S. Pat. No. 5,945,511) receptors. Such subunits can either naturallybe expressed in the cells, or be co-transfected with zcytor19 receptor.An exemplary cell system for class I cytokine receptors is to use cellsthat express gp130, and cells that co-express gp130 and LIF receptor(Gearing et al., EMBO J. 10:2839-2848, 1991; Gearing et al., U.S. Pat.No. 5,284,755). In this regard it is generally preferred to employ acell that is responsive to other cytokines that bind to receptors in thesame subfamily, such as IL-6 or LIF, because such cells will contain therequisite signal transduction pathway(s). Preferred cells of this typeinclude BaF3 cells (Palacios and Steinmetz, Cell 41: 727-734, 1985;Mathey-Prevot et al., Mol. Cell. Biol. 6: 4133-4135, 1986), the humanTF-1 cell line (ATCC number CRL-2003) and the DA-1 cell line (Branch etal., Blood 69:1782, 1987; Broudy et al., Blood 75:1622-1626, 1990). Inthe alternative, suitable host cells can be engineered to produce aβ-subunit or other cellular component needed for the desired cellularresponse. For example, the murine cell line BaF3 (Palacios andSteinmetz, Cell 41:727-734, 1985; Mathey-Prevot et al., Mol. Cell. Biol.6: 4133-4135, 1986), a baby hamster kidney (BHK) cell line, or theCTLL-2 cell line (ATCC TIB-214) can be transfected to express individualclass II subunits such as, interferon-gamma, alpha and beta chains andthe interferon-alpha/beta receptor alpha and beta chains, zcytor11(commonly owned U.S. Pat. No. 5,965,704), CRF2-4, DIRS1, zcytor7(commonly owned U.S. Pat. No. 5,945,511) receptors in addition tozcytor19. It is generally preferred to use a host cell and receptor(s)from the same species, however this approach allows cell lines to beengineered to express multiple receptor subunits from any species,thereby overcoming potential limitations arising from speciesspecificity. In the alternative, species homologs of the human receptorcDNA can be cloned and used within cell lines from the same species,such as a mouse cDNA, in the BaF3 cell line. Cell lines that aredependent upon one hematopoietic growth factor, such as IL-3, can thusbe engineered to become dependent upon a zcytor19 ligand oranti-zcytor19 antibody.

Cells expressing functional zcytor19 are used within screening assays. Avariety of suitable assays are known in the art. These assays are basedon the detection of a biological response in the target cell. One suchassay is a cell proliferation assay. Cells are cultured in the presenceor absence of a test compound, and cell proliferation is detected by,for example, measuring incorporation of tritiated thymidine or bycolorimetric assay based on the reduction or metabolic breakdown ofAlymar Blue™ (AccuMed, Chicago, Ill.) or3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)(Mosman, J. Immunol. Meth. 65: 55-63, 1983). An alternative assay formatuses cells that are further engineered to express a reporter gene. Thereporter gene is linked to a promoter element that is responsive to thereceptor-linked pathway, e.g, JAK/STAT pathway, and the assay detectsactivation of transcription of the reporter gene. A preferred promoterelement in this regard is a serum response element, SRE (see, forexample, Shaw et al., Cell 56:563-572, 1989). A preferred such reportergene is a luciferase gene (de Wet et al., Mol. Cell. Biol. 7:725, 1987).Expression of the luciferase gene is detected by luminescence usingmethods known in the art (e.g., Baumgartner et al., J. Biol. Chem.269:19094-29101, 1994; Schenborn and Goiffin, Promega Notes 41:11,1993). Luciferase assay kits are commercially available from, forexample, Promega Corp., Madison, Wis. Target cell lines of this type canbe used to screen libraries of chemicals, cell-conditioned culturemedia, fungal broths, soil samples, water samples, and the like.

A secretion trap method employing zcytor19 soluble receptor polypeptidewas used to isolate a zcytor19 ligand (Aldrich, et al, Cell 87:1161-1169, 1996), as explained in the Examples. Other methods foridentifying natural ligand for zcytor19 include mutagenizing acytokine-dependent cell line expressing zcytor19 and culturing it underconditions that select for autocrine growth. See WIPO publication WO95/21930.

As a receptor, the activity of zcytor19 polypeptide can be measured by asilicon-based biosensor microphysiometer which measures theextracellular acidification rate or proton excretion associated withreceptor binding and subsequent physiologic cellular responses. Anexemplary device is the Cytosensor™ Microphysiometer manufactured byMolecular Devices, Sunnyvale, Calif. Additional assays provided by thepresent invention include the use of hybrid receptor polypeptides. Thesehybrid polypeptides fall into two general classes. Within the firstclass, the intracellular domain of zcytor19, comprising approximatelyresidues 250 (Lys) to 491 (Arg) of SEQ ID NO:2 or residues 250 (Lys) to520 (Arg) of SEQ ID NO:19), is joined to the ligand-binding domain of asecond receptor. It is preferred that the second receptor be ahematopoietic cytokine receptor, such as mp1 receptor (Souyri et al.,Cell 63:1137-1147, 1990). The hybrid receptor will further comprise atransmembrane domain, which may be derived from either receptor. A DNAconstruct encoding the hybrid receptor is then inserted into a hostcell. Cells expressing the hybrid receptor are cultured in the presenceof a ligand for the binding domain and assayed for a response. Thissystem provides a means for analyzing signal transduction mediated byzcytor19 while using readily available ligands. This system can also beused to determine if particular cell lines are capable of responding tosignals transduced by zcytor19. A second class of hybrid receptorpolypeptides comprise the extracellular (ligand-binding)cytokine-binding domain (residues 21 (Arg) to 226 (Asn) of SEQ ID NO:2or SEQ ID NO:19), or cytokine-binding fragment (e.g., residues 21 (Arg)to 223 (Pro) of SEQ ID NO:2 or SEQ ID NO:19; SEQ ID NO:4) with acytoplasmic domain of a second receptor, preferably a cytokine receptor,and a transmembrane domain. The transmembrane domain may be derived fromeither receptor. Hybrid receptors of this second class are expressed incells known to be capable of responding to signals transduced by thesecond receptor. Together, these two classes of hybrid receptors enablethe use of a broad spectrum of cell types within receptor-based assaysystems.

Cells found to express a ligand for zcytor19 are then used to prepare acDNA library from which the ligand-encoding cDNA may be isolated asdisclosed above. The present invention thus provides, in addition tonovel receptor polypeptides, methods for cloning polypeptide ligands forthe receptors.

Agonist ligands for zcytor19, or anti-zcytor19 antibodies, may be usefulin stimulating cell-mediated immunity and for stimulating lymphocyteproliferation, such as in the treatment of infections involvingimmunosuppression, including certain viral infections. Additional usesinclude tumor suppression, where malignant transformation results intumor cells that are antigenic. Agonist ligands or anti-zcytor19antibodies could be used to induce cytotoxicity, which may be mediatedthrough activation of effector cells such as T-cells, NK (naturalkiller) cells, or LAK (lymphoid activated killer) cells, or induceddirectly through apoptotic pathways. For example, zcytor19 antibodiescould be used for stimulating cytotoxicity or ADCC on zcytor19-bearingcancer cells. Agonist ligands may also be useful in treating leukopeniasby increasing the levels of the affected cell type, and for enhancingthe regeneration of the T-cell repertoire after bone marrowtransplantation.

Antagonist ligands, compounds, soluble zcytor19 receptors, oranti-zcytor19 antibodies may find utility in the suppression of theimmune system, such as in the treatment of autoimmune diseases,including rheumatoid arthritis, multiple sclerosis, diabetes mellitis,inflammatory bowel disease, Crohn's disease, etc. Immune suppression canalso be used to reduce rejection of tissue or organ transplants andgrafts and to treat T-cell specific leukemias or lymphomas by inhibitingproliferation of the affected cell type.

The present invention contemplates the use of naked anti-zcytor19antibodies (or naked antibody fragments thereof), as well as the use ofimmunoconjugates to effect treatment of various disorders, includingB-cell malignancies and other cancers described herein wherein zcytor19is expressed. Such immunoconjugates as well as anti-zcytor19 antibodiescan be used for stimulating cytotoxicity or ADCC on zcytor19-bearingcancer cells. Immunoconjugates can be prepared using standardtechniques. For example, immunoconjugates can be produced by indirectlyconjugating a therapeutic agent to an antibody component (see, forexample, Shih et al., Int. J. Cancer 41:832-839 (1988); Shih et al.,Int. J. Cancer 46:1101-1106 (1990); and Shih et al., U.S. Pat. No.5,057,313). Briefly, one standard approach involves reacting an antibodycomponent having an oxidized carbohydrate portion with a carrier polymerthat has at least one free amine function and that is loaded with aplurality of drug, toxin, chelator, boron addends, or other therapeuticagent. This reaction results in an initial Schiff base (imine) linkage,which can be stabilized by reduction to a secondary amine to form thefinal conjugate.

The carrier polymer can be an aminodextran or polypeptide of at least 50amino acid residues, although other substantially equivalent polymercarriers can also be used. Preferably, the final immunoconjugate issoluble in an aqueous solution, such as mammalian serum, for ease ofadministration and effective targeting for use in therapy. Thus,solubilizing functions on the carrier polymer will enhance the serumsolubility of the final immunoconjugate.

In an alternative approach for producing immunoconjugates comprising apolypeptide therapeutic agent, the therapeutic agent is coupled toaminodextran by glutaraldehyde condensation or by reaction of activatedcarboxyl groups on the polypeptide with amines on the aminodextran.Chelators can be attached to an antibody component to prepareimmunoconjugates comprising radiometals or magnetic resonance enhancers.Illustrative chelators include derivatives of ethylenediaminetetraaceticacid and diethylenetriaminepentaacetic acid. Boron addends, such ascarboranes, can be attached to antibody components by conventionalmethods.

Immunoconjugates can also be prepared by directly conjugating anantibody component with a therapeutic agent. The general procedure isanalogous to the indirect method of conjugation except that atherapeutic agent is directly attached to an oxidized antibodycomponent.

As a further illustration, a therapeutic agent can be attached at thehinge region of a reduced antibody component via disulfide bondformation. For example, the tetanus toxoid peptides can be constructedwith a single cysteine residue that is used to attach the peptide to anantibody component. As an alternative, such peptides can be attached tothe antibody component using a heterobifunctional cross-linker, such asN-succinyl 3-(2-pyridyldithio)proprionate. Yu et al., Int. J. Cancer56:244 (1994). General techniques for such conjugation are well-known inthe art. See, for example, Wong, Chemistry Of Protein Conjugation AndCross-Linking (CRC Press 1991); Upeslacis et al., “Modification ofAntibodies by Chemical Methods,” in Monoclonal Antibodies: PrinciplesAnd Applications, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc.1995); Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in Monoclonal Antibodies: Production,Engineering And Clinical Application, Ritter et al. (eds.), pages 60-84(Cambridge University Press 1995).

As described above, carbohydrate moieties in the Fc region of anantibody can be used to conjugate a therapeutic agent. However, the Fcregion is absent if an antibody fragment is used as the antibodycomponent of the immunoconjugate. Nevertheless, it is possible tointroduce a carbohydrate moiety into the light chain variable region ofan antibody or antibody fragment. See, for example, Leung et al., J.Immunol. 154:5919 (1995); Hansen et al., U.S. Pat. No. 5,443,953 (1995).The engineered carbohydrate moiety is then used to attach a therapeuticagent.

In addition, those of skill in the art will recognize numerous possiblevariations of the conjugation methods. For example, the carbohydratemoiety can be used to attach polyethyleneglycol in order to extend thehalf-life of an intact antibody, or antigen-binding fragment thereof, inblood, lymph, or other extracellular fluids. Moreover, it is possible toconstruct a divalent immunoconjugate by attaching therapeutic agents toa carbohydrate moiety and to a free sulfhydryl group. Such a freesulfhydryl group may be located in the hinge region of the antibodycomponent.

One type of immunoconjugate comprises an antibody component and apolypeptide cytotoxin. An example of a suitable polypeptide cytotoxin isa ribosome-inactivating protein. Type I ribosome-inactivating proteinsare single-chain proteins, while type II ribosome-inactivating proteinsconsist of two nonidentical subunits (A and B chains) joined by adisulfide bond (for a review, see Soria et al., Targeted Diagn. Ther.7:193 (1992)). Useful type I ribosome-inactivating proteins includepolypeptides from Saponaria officinalis (e.g., saporin-1, saporin-2,saporin-3, saporin-6), Momordica charantia (e.g, momordin), Byroniadioica (e.g., bryodin, bryodin-2), Trichosanthes kirilowii (e.g.,trichosanthin, trichokirin), Gelonium mulfiflorum (e.g., gelonin),Phytolacca americana (e.g., pokeweed antiviral protein, pokeweedantiviral protein-II, pokeweed antiviral protein-S), Phytolaccadodecandra (e.g., dodecandrin, Mirabilis antiviral protein), and thelike. Ribosome-inactivating proteins are described, for example, byWalsh et al., U.S. Pat. No. 5,635,384.

Suitable type II ribosome-inactivating proteins include polypeptidesfrom Ricinus communis (e.g., ricin), Abrus precatorius (e.g., abrin),Adenia digitata (e.g., modeccin), and the like. Since type IIribosome-inactiving proteins include a B chain that binds galactosidesand a toxic A chain that depurinates adensoine, type IIribosome-inactivating protein conjugates should include the A chain.Additional useful ribosome-inactivating proteins include bouganin,clavin, maize ribosome-inactivating proteins, Vaccaria pyramidataribosome-inactivating proteins, nigrine b, basic nigrine 1, ebuline,racemosine b, luffin-b, luffin-S, and other ribosome-inactivatingproteins known to those of skill in the art. See, for example, Bolognesiand Stirpe, international publication No. WO98/55623, Colnaghi et al.,international publication No. WO97/49726, Hey et al., U.S. Pat. No.5,635,384, Bolognesi and Stirpe, international publication No.WO95/07297, Arias et al., international publication No. WO94/20540,Watanabe et al., J. Biochem. 106:6 977 (1989); Islam et al., Agric.Biol. Chem. 55:229 (1991), and Gao et al., FEBS Lett. 347:257 (1994).

Analogs and variants of naturally-occurring ribosome-inactivatingproteins are also suitable for the targeting compositions describedherein, and such proteins are known to those of skill in the art.Ribosome-inactivating proteins can be produced using publicly availableamino acid and nucleotide sequences. As an illustration, a nucleotidesequence encoding saporin-6 is disclosed by Lorenzetti et al., U.S. Pat.No. 5,529,932, while Walsh et al., U.S. Pat. No. 5,635,384, describemaize and barley ribosome-inactivating protein nucleotide and amino acidsequences. Moreover, ribosome-inactivating proteins are alsocommercially available.

Additional polypeptide cytotoxins include ribonuclease, DNase I,Staphylococcal enterotoxin-A, diphtheria toxin, Pseudomonas exotoxin,and Pseudomonas endotoxin. See, for example, Pastan et al., Cell 47:641(1986), and Goldenberg, C A—A Cancer Journal for Clinicians 44:43(1994).

Another general type of useful cytotoxin is a tyrosine kinase inhibitor.Since the activation of proliferation by tyrosine kinases has beensuggested to play a role in the development and progression of tumors,this activation can be inhibited by anti-zcytor19 antibody componentsthat deliver tyrosine kinase inhibitors. Suitable tyrosine kinaseinhibitors include isoflavones, such as genistein(5,7,4′-trihydroxyisoflavone), daidzein (7,4″-dihydroxyisoflavone), andbiochanin A (4-methoxygenistein), and the like. Methods of conjugatingtyrosine inhibitors to a growth factor are described, for example, byUckun, U.S. Pat. No. 5,911,995.

Another group of useful polypeptide cytotoxins includesimmunomodulators. As used herein, the term “immunomodulator” includescytokines, stem cell growth factors, lymphotoxins, co-stimulatorymolecules, hematopoietic factors, and the like, as well as syntheticanalogs of these molecules. Examples of immunomodulators include tumornecrosis factor, interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14,IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-28A, IL-28B,and IL-29), colony stimulating factors (e.g., granulocyte-colonystimulating factor and granulocyte macrophage-colony stimulatingfactor), interferons (e.g., interferons-α, -β, -γ, -ω, -ε, and -τ), thestem cell growth factor designated “S1 factor,” erythropoietin, andthrombopoietin. Illustrative immunomodulator moieties include IL-2,IL-6, IL-10, interferon-□, TNF-□, and the like.

Immunoconjugates that include an immunomodulator provide a means todeliver an immunomodulator to a target cell, and are particularly usefulagainst tumor cells. The cytotoxic effects of immunomodulators are wellknown to those of skill in the art. See, for example, Klegerman et al.,“Lymphokines and Monokines,” in Biotechnology And Pharmacy, Pessuto etal. (eds.), pages 53-70 (Chapman & Hall 1993). As an illustration,interferons can inhibit cell proliferation by inducing increasedexpression of class I histocompatibility antigens on the surface ofvarious cells and thus, enhance the rate of destruction of cells bycytotoxic T lymphocytes. Furthermore, tumor necrosis factors, such astumor necrosis factor-α, are believed to produce cytotoxic effects byinducing DNA fragmentation.

The present invention also includes immunoconjugates that comprise anucleic acid molecule encoding a cytotoxin. As an example of thisapproach, Hoganson et al., Human Gene Ther. 9:2565 (1998), describeFGF-2 mediated delivery of a saporin gene by producing anFGF-2-polylysine conjugate which was condensed with an expression vectorcomprising a saporin gene. Other suitable toxins are known to those ofskill in the art.

Conjugates of cytotoxic polypeptides and antibody components can beprepared using standard techniques for conjugating polypeptides. Forexample, Lain and Kelleher, U.S. Pat. No. 5,055,291, describe theproduction of antibodies conjugated with either diphtheria toxinfragment A or ricin toxin. The general approach is also illustrated bymethods of conjugating fibroblast growth factor with saporin, asdescribed by Lappi et al., Biochem. Biophys. Res. Commun. 160:917(1989), Soria et al., Targeted Diagn. Ther. 7:193 (1992), Buechler etal., Eur. J. Biochem. 234:706 (1995), Behar-Cohen et al., Invest.Ophthalmol. Vis. Sci. 36:2434 (1995), Lappi and Baird, U.S. Pat. No.5,191,067, Calabresi et al., U.S. Pat. No. 5,478,804, and Lappi andBaird, U.S. Pat. No. 5,576,288. Also see, Ghetie and Vitteta, “ChemicalConstruction of Immunotoxins,” in Drug Targeting: Strategies,Principles, and Applications, Francis and Delgado (Eds.), pages 1-26(Humana Press, Inc. 2000), Hall (Ed.), Immunotoxin Methods and Protocols(Humana Press, Inc. 2000), and Newton and Rybak, “Construction ofRibonuclease-Antibody Conjugates for Selective Cytotoxicity,” in DrugTargeting: Strategies, Principles, and Applications, Francis and Delgado(Eds.), pages 27-35 (Humana Press, Inc. 2000).

Alternatively, fusion proteins comprising an antibody component and acytotoxic polypeptide can be produced using standard methods. Methods ofpreparing fusion proteins comprising a cytotoxic polypeptide moiety arewell-known in the art of antibody-toxin fusion protein production. Forexample, antibody fusion proteins comprising an interleukin-2 moiety aredescribed by Boleti et al, Ann. Oncol. 6:945 (1995), Nicolet et al.,Cancer Gene Ther. 2:161 (1995), Becker et al., Proc. Nat'l Acad. Sci.USA 93:7826 (1996), Hank et al., Clin. Cancer Res. 2:1951 (1996), and Huet al., Cancer Res. 56:4998 (1996). In addition, Yang et al., Hum.Antibodies Hybridomas 6:129 (1995), describe a fusion protein thatincludes an F(ab′)₂ fragment and a tumor necrosis factor alpha moiety.Antibody-Pseudomonas exotoxin A fusion proteins have been described byChaudhary et al., Nature 339:394 (1989), Brinkmann et al., Proc. Nat'lAcad. Sci. USA 88:8616 (1991), Batra et al, Proc. Nat'l Acad. Sci. USA89:5867 (1992), Friedman et al., J. Immunol 150:3054 (1993), Wels et al,Int. J. Can. 60:137 (1995), Fominaya et al., J. Biol. Chem. 271:10560(1996), Kuan et al., Biochemistry 35:2872 (1996), and Schmidt et al.,Int. J. Can. 65:538 (1996). Antibody-toxin fusion proteins containing adiphtheria toxin moiety have been described by Kreitman et al., Leukemia7:553 (1993), Nicholls et al., J. Biol. Chem. 268:5302 (1993), Thompsonet al., J. Biol. Chem. 270:28037 (1995), and Vallera et al., Blood88:2342 (1996). Deonarain et al., Tumor Targeting 1:177 (1995), havedescribed an antibody-toxin fusion protein having an RNase moiety, whileLinardou et al., Cell Biophys. 24-25:243 (1994), produced anantibody-toxin fusion protein comprising a DNase I component. Geloninwas used as the toxin moiety in the antibody-toxin fusion protein ofBetter et al., J. Biol. Chem. 270:14951 (1995). As a further example,Dohlsten et al., Proc. Nat'l Acad. Sci. USA 91:8945 (1994), reported anantibody-toxin fusion protein comprising Staphylococcal enterotoxin-A.Also see, Newton and Rybak, “Preparation of Recombinant RNaseSingle-Chain Antibody Fusion Proteins,” in Drug Targeting: Strategies,Principles, and Applications, Francis and Delgado (Eds.), pages 77-95(Humana Press, Inc. 2000).

As an alternative to a polypeptide cytotoxin, immunoconjugates cancomprise a radioisotope as the cytotoxic moiety. For example, animmunoconjugate can comprise an anti-zcytor19 antibody component and anα-emitting radioisotope, a β-emitting radioisotope, a γ-emittingradioisotope, an Auger electron emitter, a neutron capturing agent thatemits α-particles or a radioisotope that decays by electron capture.Suitable radioisotopes include ¹⁹⁸Au, ¹⁹⁹Au, ³²P, ³³P, ¹²⁵I, ¹³¹I, ¹²³I,⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ⁶⁷Cu, ²¹¹At, ⁴⁷Sc, ¹⁰³Pb, ¹⁰⁹Pd, ²¹²Pb, ⁷¹Ge, ⁷⁷As,¹⁰⁵Rh, ¹¹³Ag, ¹¹⁹Sb, ¹²¹Sn, ¹³¹Cs, ¹⁴³Pr, ¹⁶¹Tb, ¹⁷⁷Lu, ¹⁹¹Os,^(193M)Pt, ¹⁹⁷Hg, and the like.

A radioisotope can be attached to an antibody component directly orindirectly, via a chelating agent. For example, ⁶⁷Cu, which providesβ-particles and γ-rays, can be conjugated to an antibody component usingthe chelating agent, p-bromoacetamido-benzyl-tetraethylaminetetraaceticacid. Chase and Shapiro, “Medical Applications of Radioisotopes,” inGennaro (Ed.), Remington: The Science and Practice of Pharmacy, 19thEdition, pages 843-865 (Mack Publishing Company 1995). As analternative, ⁹⁰Y, which emits an energetic β-particle, can be coupled toan antibody component using diethylenetriaminepentaacetic acid.Moreover, an exemplary suitable method for the direct radiolabeling ofan antibody component with ¹³¹I is described by Stein et al., AntibodyImmunoconj. Radiopharm. 4:703 (1991). Alternatively, boron addends suchas carboranes can be attached to antibody components, using standardtechniques.

Another type of suitable cytotoxin for the preparation ofimmunoconjugates is a chemotherapeutic drug. Illustrativechemotherapeutic drugs include nitrogen mustards, alkyl sulfonates,nitrosoureas, triazenes, folic acid analogs, pyrimidine analogs, purineanalogs, antibiotics, epipodophyllotoxins, platinum coordinationcomplexes, and the like. Specific examples of chemotherapeutic drugsinclude methotrexate, doxorubicin, daunorubicin, cytosinarabinoside,cis-platin, vindesine, mitomycin, bleomycin, melphalan, chlorambucil,maytansinoids, calicheamicin, taxol, and the like. Suitablechemotherapeutic agents are described in Remington: The Science andPractice of Pharmacy, 19th Edition (Mack Publishing Co. 1995), and inGoodman And Gilman's The Pharmacological Basis Of Therapeutics, 9th Ed.(MacMillan Publishing Co. 1995). Other suitable chemotherapeutic agentsare known to those of skill in the art.

In another approach, immunoconjugates are prepared by conjugatingphotoactive agents or dyes to an antibody component. Fluorescent andother chromogens, or dyes, such as porphyrins sensitive to visiblelight, have been used to detect and to treat lesions by directing thesuitable light to the lesion. This type of “photoradiation,”“phototherapy,” or “photodynamic” therapy is described, for example, byMew et al., J. Immunol. 130:1473 (1983), Jori et al. (eds.),Photodynamic Therapy Of Tumors And Other Diseases (Libreria Progetto1985), Oseroff et al., Proc. Natl. Acad. Sci. USA 83:8744 (1986), vanden Bergh, Chem. Britain 22:430 (1986), Hasan et al., Prog. Clin. Biol.Res. 288:471 (1989), Tatsuta et al., Lasers Surg. Med. 9:422 (1989), andPelegrin et al., Cancer 67:2529 (1991).

The approaches described above can also be used to prepare multispecificantibody compositions that comprise an immunoconjugate.

Antibodies disclosed herein include antibodies that bind thezcytor19/CRF2-4 heterodimeric complex, including the heterodimericsoluble receptor.

Anti-zcytor19 antibodies, and multispecific antibody compositions can beused to modulate the immune system by preventing the binding of zcytor19ligands (for example, zcyto20, zcyto21, zcyto22, zcyto24, and zcyto25)with endogenous zcytor19 receptors. Such antibodies can be administeredto any subject in need of treatment, and the present inventioncontemplates both veterinary and human therapeutic uses. Illustrativesubjects include mammalian subjects, such as farm animals, domesticanimals, and human patients.

Multispecific antibody compositions and dual reactive antibodies thatbind zcytor19 can be used for the treatment of autoimmune diseases, Bcell cancers, immunomodulation, and other pathologies (e.g., ITCP, Tcell-mediated diseases, cattleman's disease, autoimmune disease,myelodysplastic syndrome, and the like), renal diseases, graftrejection, and graft versus host disease. The antibodies of the presentinvention can be targeted to specifically regulate B cell responsesduring the immune response. Additionally, the antibodies of the presentinvention can be used to modulate B cell development, antigenpresentation by B cells, antibody production, and cytokine production.

Antagonistic anti-zcytor19 antibodies can be useful to neutralize theeffects of zcytor19 ligands for treating B cell lymphomas and leukemias,chronic or acute lymphocytic leukemia, myelomas such as multiplemyeloma, plasma cytomas, and lymphomas such as non-Hodgkins lymphoma,for which an increase in zcytor19 ligand polypeptides is associated, orwhere zcytor19 ligand is a survival factor or growth factor.Anti-zcytor19 antibodies can also be used to treat Epstein Barrvirus-associated lymphomas arising in immunocompromised patients (e.g,AIDS or organ transplant).

Anti-zcytor19 antibodies that induce a signal by binding with zcytor19may inhibit the growth of lymphoma and leukemia cells directly viainduction of signals that lead to growth inhibition, cell cycle arrest,apoptosis, or tumor cell death. Zcytor19 antibodies that initiate asignal are preferred antibodies to directly inhibit or kill cancercells. In addition, agonistic anti-zcytor19 monoclonal antibodies mayactivate normal B cells and promote an anticancer immune response.Anti-zcytor19 antibodies may directly inhibit the growth of leukemias,lymphomas, and multiple myelomas, and the antibodies may engage immuneeffector functions. Anti-zcytor19 monoclonal antibodies may enableantibody-dependent cellular cytotoxicity, complement dependentcytotoxicity, and phagocytosis.

Zcytor19 ligand may be expressed in neutrophils, monocytes, dendriticcells, and activated monocytes. In certain autoimmune disorders (e.g.,myasthenia gravis, and rheumatoid arthritis), B cells might exacerbateautoimmunity after activation by zcytor19 ligand. Immunosuppressantproteins that selectively block the action of B-lymphocytes would be ofuse in treating disease. Autoantibody production is common to severalautoimmune diseases and contributes to tissue destruction andexacerbation of disease. Autoantibodies can also lead to the occurrenceof immune complex deposition complications and lead to many symptoms ofsystemic lupus erythematosus, including kidney failure, neuralgicsymptoms and death. Modulating antibody production independent ofcellular response would also be beneficial in many disease states. Bcells have also been shown to play a role in the secretion ofarthritogenic immunoglobulins in rheumatoid arthritis. As such,inhibition of zcytor19 ligand antibody production would be beneficial intreatment of autoimmune diseases such as myasthenia gravis andrheumatoid arthritis. Immunosuppressant therapeutics such asanti-zcytor19 antibodies that selectively block or neutralize the actionof B-lymphocytes would be useful for such purposes.

The invention provides methods employing anti-zcytor19 antibodies, ormultispecific antibody compositions, for selectively blocking orneutralizing the actions of B-cells in association with end stage renaldiseases, which may or may not be associated with autoimmune diseases.Such methods would also be useful for treating immunologic renaldiseases. Such methods would be would be useful for treatingglomerulonephritis associated with diseases such as membranousnephropathy, IgA nephropathy or Berger's Disease, IgM nephropathy,Goodpasture's Disease, post-infectious glomerulonephritis,mesangioproliferative disease, chronic lymphocytic leukemia,minimal-change nephrotic syndrome. Such methods would also serve astherapeutic applications for treating secondary glomerulonephritis orvasculitis associated with such diseases as lupus, polyarteritis,Henoch-Schonlein, Scleroderma, HIV-related diseases, amyloidosis orhemolytic uremic syndrome. The methods of the present invention wouldalso be useful as part of a therapeutic application for treatinginterstitial nephritis or pyelonephritis associated with chronicpyelonephritis, analgesic abuse, nephrocalcinosis, nephropathy caused byother agents, nephrolithiasis, or chronic or acute interstitialnephritis.

Additionally, the invention provides methods employing anti-zcytor19antibodies, or multispecific antibody compositions, for selectivelyblocking or neutralizing the viral infection associated with the liver.As shown in Example 24, while normal and diseased liver specimens showexpression of zcytoR19 mRNA, there is specific expression of thereceptor in liver specimens that are positive for Hepatitis Virus C, andHepatitis B.

When liver disease is inflammatory and continuing for at least sixmonths, it is generally considered chronic hepatitis. Hepatitis C virus(HCV) patients actively infected will be positive for HCV-RNA in theirblood, which is detectable by reverse transcritptase/polymerase chainreaction (RT-PCR) assays. The methods of the present invention will slowthe progression of the liver disease, and can be measured, for example,as improved serum alanine transaminase (ALT) levels, improved levels ofaspartate aminotrasnferase (AST), decreased portal inflammation asdetermined by biopsy, or decrease in hepatocytic necrosis. Histologicalimprovement can be measured using the Histological Activity Index (Daviset al., New Eng. J. Of Med. 321:1501-1506, 1989; Knodell et al.,Hepatology 1:431-435, 1981). Other means for measuring improvement areknown in the art, and will be determined by the clinician, and caninclude, for example, evaluation of HCV antibodies (Kuo, et al. Science,244:362-364, 1989).

The present invention also provides methods for treatment of renal orurological neoplasms, multiple myelomas, lymphomas, leukemias, lightchain neuropathy, or amyloidosis.

The invention also provides methods for blocking or inhibiting activatedB cells using anti-zcytor19 antibodies, or multispecific antibodycompositions, for the treatment of asthma and other chronic airwaydiseases such as bronchitis and emphysema.

Also provided are methods for inhibiting or neutralizing a T cellresponse using anti-zcytor19 antibodies, or multispecific antibodycompositions, for immunosuppression, in particular for such therapeuticuse as for graft-versus-host disease and graft rejection. Moreover,anti-zcytor19 antibodies, or multispecific antibody compositions, wouldbe useful in therapeutic protocols for treatment of such autoimmunediseases as insulin dependent diabetes mellitus (IDDM), multiplesclerosis, rheumatoid arthritis, systemic lupus erythematosus,inflammatory bowel disease (IBD), and Crohn's Disease. Methods of thepresent invention would have additional therapeutic value for treatingchronic inflammatory diseases, in particular to lessen joint pain,swelling, anemia and other associated symptoms as well as treatingseptic shock.

B cell responses are important in fighting infectious diseases includingbacterial, viral, protozoan and parasitic infections. Antibodies againstinfectious microorganisms can immobilize the pathogen by binding toantigen followed by complement mediated lysis or cell mediated attack.Agonistic, or signaling, anti-zcytor19 antibodies may serve to boost thehumoral response and would be a useful therapeutic for individuals atrisk for an infectious disease or as a supplement to vaccination.

Well established animal models are available to test in vivo efficacy ofanti-zcytor19 antibodies, or multispecific antibody compositions, of thepresent invention in certain disease states. As an illustration,anti-zcytor19 antibodies can be tested in vivo in a number of animalmodels of autoimmune disease, such as MRL-lpr/lpr or NZB×NZW F1 congenicmouse strains which serve as a model of systemic lupus erythematosus.Such animal models are known in the art.

Generally, the dosage of administered anti-zcytor19 antibodies, ormultispecific antibody compositions, will vary depending upon suchfactors as the subject's age, weight, height, sex, general medicalcondition and previous medical history. As an illustration,anti-zcytor19 antibodies, or multispecific antibody compositions, can beadministered at low protein doses, such as 20 to 100 milligrams proteinper dose, given once, or repeatedly. Alternatively, anti-zcytor19antibodies, or multispecific antibody compositions, can be administeredin doses of 30 to 90 milligrams protein per dose, or 40 to 80 milligramsprotein per dose, or 50 to 70 milligrams protein per dose, although alower or higher dosage also may be administered as circumstancesdictate.

Administration of antibody components to a subject can be intravenous,intraarterial, intraperitoneal, intramuscular, subcutaneous,intrapleural, intrathecal, by perfusion through a regional catheter, orby direct intralesional injection. When administering therapeuticproteins by injection, the administration may be by continuous infusionor by single or multiple boluses. Additional routes of administrationinclude oral, mucosal-membrane, pulmonary, and transcutaneous.

A pharmaceutical composition comprising an anti-zcytor19 antibody, orbispecific antibody components, can be formulated according to knownmethods to prepare pharmaceutically useful compositions, whereby thetherapeutic proteins are combined in a mixture with a pharmaceuticallyacceptable carrier. A composition is said to be a “pharmaceuticallyacceptable carrier” if its administration can be tolerated by arecipient patient. Sterile phosphate-buffered saline is one example of apharmaceutically acceptable carrier. Other suitable carriers arewell-known to those in the art. See, for example, Gennaro (ed.),Remington's Pharmaceutical Sciences, 19th Edition (Mack PublishingCompany 1995).

For purposes of therapy, anti-zcytor19 antibodies, or bispecificantibody components, and a pharmaceutically acceptable carrier areadministered to a patient in a therapeutically effective amount. Acombination of anti-zcytor19 antibodies, or bispecific antibodycomponents, and a pharmaceutically acceptable carrier is said to beadministered in a “therapeutically effective amount” if the amountadministered is physiologically significant. An agent is physiologicallysignificant if its presence results in a detectable change in thephysiology of a recipient patient. For example, an agent used to treatinflammation is physiologically significant if its presence alleviatesthe inflammatory response. As another example, an agent used to inhibitthe growth of tumor cells is physiologically significant if theadministration of the agent results in a decrease in the number of tumorcells, decreased metastasis, a decrease in the size of a solid tumor, orincreased necrosis of a tumor.

A pharmaceutical composition comprising anti-zcytor19 antibodies, orbispecific antibody components, can be furnished in liquid form, in anaerosol, or in solid form. Liquid forms, are illustrated by injectablesolutions and oral suspensions. Exemplary solid forms include capsules,tablets, and controlled-release forms. The latter form is illustrated byminiosmotic pumps and implants (Bremer et al., Pharm. Biotechnol. 10:239(1997); Ranade, “Implants in Drug Delivery,” in Drug Delivery Systems,Ranade and Hollinger (eds.), pages 95-123 (CRC Press 1995); Bremer etal., “Protein Delivery with Infusion Pumps,” in Protein Delivery:Physical Systems, Sanders and Hendren (eds.), pages 239-254 (PlenumPress 1997); Yewey et al., “Delivery of Proteins from a ControlledRelease Injectable Implant,” in Protein Delivery: Physical Systems,Sanders and Hendren (eds.), pages 93-117 (Plenum Press 1997)).

As another example, liposomes provide a means to deliver anti-zcytor19antibodies, or bispecific antibody components, to a subjectintravenously, intraperitoneally, intrathecally, intramuscularly,subcutaneously, or via oral administration, inhalation, or intranasaladministration. Liposomes are microscopic vesicles that consist of oneor more lipid bilayers surrounding aqueous compartments (see, generally,Bakker-Woudenberg et al., Eur. J. Clin. Microbial. Infect. Dis. 12(Suppl. 1):S61 (1993), Kim, Drugs 46:618 (1993), and Ranade,“Site-Specific Drug Delivery Using Liposomes as Carriers,” in DrugDelivery Systems, Ranade and Hollinger (Eds.), pages 3-24 (CRC Press1995)). Liposomes are similar in composition to cellular membranes andas a result, liposomes can be administered safely and are biodegradable.Depending on the method of preparation, liposomes may be unilamellar ormultilamellar, and liposomes can vary in size with diameters rangingfrom 0.02 μm to greater than 10 μm. A variety of agents can beencapsulated in liposomes: hydrophobic agents partition in the bilayersand hydrophilic agents partition within the inner aqueous space(s) (see,for example, Machy et al., Liposomes In Cell Biology And Pharmacology(John Libbey 1987), and Ostro et al., American J. Hosp. Pharm. 46:1576(1989)). Moreover, it is possible to control the therapeuticavailability of the encapsulated agent by varying liposome size, thenumber of bilayers, lipid composition, as well as the charge and surfacecharacteristics of the liposomes.

As an alternative to administering liposomes that comprise ananti-zcytor19 antibody component, target cells can be prelabeled withbiotinylated anti-zcytor19 antibodies. After plasma elimination of freeantibody, streptavidin-conjugated liposomes are administered. Thisgeneral approach is described, for example, by Harasym et al., Adv. DrugDeliv. Rev. 32:99 (1998). Such an approach can also be used to preparemultispecific antibody compositions.

The present invention also contemplates chemically modified antibodycomponents, in which an antibody component is linked with a polymer.Typically, the polymer is water soluble so that an antibody componentdoes not precipitate in an aqueous environment, such as a physiologicalenvironment. An example of a suitable polymer is one that has beenmodified to have a single reactive group, such as an active ester foracylation, or an aldehyde for alkylation. In this way, the degree ofpolymerization can be controlled. An example of a reactive aldehyde ispolyethylene glycol propionaldehyde, or mono-(C₁-C₁₀) alkoxy, or aryloxyderivatives thereof (see, for example, Harris, et al., U.S. Pat. No.5,252,714). The polymer may be branched or unbranched. Moreover, amixture of polymers can be used to produce conjugates with antibodycomponents.

Suitable water-soluble polymers include polyethylene glycol (PEG),monomethoxy-PEG, mono-(C₁-C₁₀)alkoxy-PEG, aryloxy-PEG, poly-(N-vinylpyrrolidone)PEG, tresyl monomethoxy PEG, PEG propionaldehyde,bis-succinimidyl carbonate PEG, propylene glycol homopolymers, apolypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols(e.g., glycerol), polyvinyl alcohol, dextran, cellulose, or othercarbohydrate-based polymers. Suitable PEG may have a molecular weightfrom about 600 to about 60,000, including, for example, 5,000, 12,000,20,000 and 25,000. A conjugate can also comprise a mixture of suchwater-soluble polymers.

General methods for producing conjugates comprising a polypeptide andwater-soluble polymer moieties are known in the art. See, for example,Karasiewicz et al., U.S. Pat. No. 5,382,657, Greenwald et al., U.S. Pat.No. 5,738,846, Nieforth et al., Clin. Pharmacol. Ther. 59:636 (1996),Monkarsh et al., Anal. Biochem. 247:434 (1997)).

Polypeptide cytotoxins can also be conjugated with a soluble polymerusing the above methods either before or after conjugation to anantibody component. Soluble polymers can also be conjugated withantibody fusion proteins.

Naked anti-zcytor19 antibodies, or antibody fragments, can besupplemented with immunoconjugate or antibody fusion proteinadministration. In one variation, naked anti-zcytor19 antibodies (ornaked antibody fragments) are administered with low-dose radiolabeledanti-zcytor19 antibodies or antibody fragments. As a second alternative,naked anti-zcytor19 antibodies (or antibody fragments) are administeredwith low-dose radiolabeled anti-zcytor19 antibodies-cytokineimmunoconjugates. As a third alternative, naked anti-zcytor19 antibodies(or antibody fragments) are administered with anti-zcytor19-cytokineimmunoconjugates that are not radiolabeled. With regard to “low doses”of ¹³¹I-labeled immunoconjugates, a preferable dosage is in the range of15 to 40 mCi, while the most preferable range is 20 to 30 mCi. Incontrast, a preferred dosage of ⁹⁰Y-labeled immunoconjugates is in therange from 10 to 30 mCi, while the most preferable range is 10 to 20mCi. Similarly, bispecific antibody components can be supplemented withimmunoconjugate or antibody fusion protein administration.

immunoconjugates having a boron addend-loaded carrier for thermalneutron activation therapy will normally be effected in similar ways.However, it will be advantageous to wait until non-targetedimmunoconjugate clears before neutron irradiation is performed.Clearance can be accelerated using an antibody that binds to theimmunoconjugate. See U.S. Pat. No. 4,624,846 for a description of thisgeneral principle.

The present invention also contemplates a method of treatment in whichimmunomodulators are administered to prevent, mitigate or reverseradiation-induced or drug-induced toxicity of normal cells, andespecially hematopoietic cells. Adjunct immunomodulator therapy allowsthe administration of higher doses of cytotoxic agents due to increasedtolerance of the recipient mammal. Moreover, adjunct immunomodulatortherapy can prevent, palliate, or reverse dose-limiting marrow toxicity.Examples of suitable immunomodulators for adjunct therapy includegranulocyte-colony stimulating factor, granulocyte macrophage-colonystimulating factor, thrombopoietin, IL-1, IL-3, IL-12, and the like. Themethod of adjunct immunomodulator therapy is disclosed by Goldenberg,U.S. Pat. No. 5,120,525.

The efficacy of anti-zcytor19 antibody therapy can be enhanced bysupplementing naked antibody components with immunoconjugates and otherforms of supplemental therapy described herein. In such multimodalregimens, the supplemental therapeutic compositions can be administeredbefore, concurrently or after administration of naked anti-zcytor19antibodies. Multimodal therapies of the present invention furtherinclude immunotherapy with naked anti-zcytor19 antibody componentssupplemented with administration of anti-zcytor19 immunoconjugates. Inanother form of multimodal therapy, subjects receive naked anti-zcytor19antibodies and standard cancer chemotherapy.

The antibodies and antibody fragments of the present invention can beused as vaccines to treat the various disorders and diseases describedabove. As an example, an antibody component of a dual reactive zcytor19receptor monoclonal antibody can provide a suitable basis for a vaccine.Cysteine-rich regions of zcytor19 receptors can also provide usefulcomponents for a vaccine. For example, a vaccine can comprise at leastone of the following polypeptides: a polypeptide comprising amino acidresidues 8 to 41 of SEQ ID NO:2, a polypeptide comprising amino acidresidues 34 to 66 of SEQ ID NO:4, and a polypeptide comprising aminoacid residues 71 to 104 of SEQ ID NO:4.

Pharmaceutical compositions may be supplied as a kit comprising acontainer that comprises anti-zcytor19 antibody components, orbispecific antibody components. Therapeutic molecules can be provided inthe form of an injectable solution for single or multiple doses, or as asterile powder that will be reconstituted before injection.Alternatively, such a kit can include a dry-powder disperser, liquidaerosol generator, or nebulizer for administration of an anti-zcytor19antibody component. Such a kit may further comprise written informationon indications and usage of the pharmaceutical composition. Moreover,such information may include a statement that the composition iscontraindicated in patients with known hypersensitivity to exogenousantibodies.

Zcytor19 polypeptides, such as soluble zcytor19 receptors, may also beused within diagnostic systems for the detection of circulating levelsof ligand. Within a related embodiment, antibodies or other agents thatspecifically bind to zcytor19 receptor polypeptides can be used todetect circulating receptor polypeptides. Elevated or depressed levelsof ligand or receptor polypeptides may be indicative of pathologicalconditions, including cancer. Soluble receptor polypeptides maycontribute to pathologic processes and can be an indirect marker of anunderlying disease. For example, elevated levels of soluble IL-2receptor in human serum have been associated with a wide variety ofinflammatory and neoplastic conditions, such as myocardial infarction,asthma, myasthenia gravis, rheumatoid arthritis, acute T-cell leukemia,B-cell lymphomas, chronic lymphocytic leukemia, colon cancer, breastcancer, and ovarian cancer (Heaney et al., Blood 87:847-857, 1996).Similarly, as zcytor19 is expressed in B-cell leukemia cells, anincrease of zcytor19 expression can even serve as a marker of anunderlying disease, such as leukemia.

A ligand-binding polypeptide of a zcytor19 receptor, or “solublereceptor,” can be prepared by expressing a truncated DNA encoding thezcytor19 extracellular cytokine-binding domain (residues 21 (Arg) to 226(Asn) of SEQ ID NO:2 or SEQ ID NO:19), cytokine-binding fragment (e.g.,residues 21 (Arg) to 223 (Pro) of SEQ ID NO:2 or SEQ ID NO:19; SEQ IDNO:4), the soluble version of zcytor19 variant, or the correspondingregion of a non-human receptor. It is preferred that the extracellulardomain be prepared in a form substantially free of transmembrane andintracellular polypeptide segments. Moreover, ligand-binding polypeptidefragments within the zcytor19 cytokine-binding domain, described above,can also serve as zcytor19 soluble receptors for uses described herein.To direct the export of a receptor polypeptide from the host cell, thereceptor DNA is linked to a second DNA segment encoding a secretorypeptide, such as a t-PA secretory peptide or a zcytor19 secretorypeptide. To facilitate purification of the secreted receptorpolypeptide, a C-terminal extension, such as a poly-histidine tag,Glu-Glu tag peptide, substance P, Flag™ peptide (Hopp et al.,Bio/Technology 6:1204-1210, 1988; available from Eastman Kodak Co., NewHaven, Conn.) or another polypeptide or protein for which an antibody orother specific binding agent is available, can be fused to the receptorpolypeptide.

In an alternative approach, a receptor extracellular domain can beexpressed as a fusion with immunoglobulin heavy chain constant regions,typically an F_(c) fragment, which contains two constant region domainsand lacks the variable region. Such fusions are typically secreted asmultimeric molecules wherein the Fc portions are disulfide bonded toeach other and two receptor polypeptides are arrayed in close proximityto each other. Fusions of this type can be used to affinity purify thecognate ligand from solution, as an in vitro assay tool, to blocksignals in vitro by specifically titrating out ligand, and asantagonists in vivo by administering them parenterally to bindcirculating ligand and clear it from the circulation. To purify ligand,a zcytor19-Ig chimera is added to a sample containing the ligand (e.g.,cell-conditioned culture media or tissue extracts) under conditions thatfacilitate receptor-ligand binding (typically near-physiologicaltemperature, pH, and ionic strength). The chimera-ligand complex is thenseparated by the mixture using protein A, which is immobilized on asolid support (e.g., insoluble resin beads). The ligand is then elutedusing conventional chemical techniques, such as with a salt or pHgradient. In the alternative, the chimera itself can be bound to a solidsupport, with binding and elution carried out as above. Collectedfractions can be re-fractionated until the desired level of purity isreached.

Moreover, zcytor19 soluble receptors can be used as a “ligand sink,”i.e., antagonist, to bind ligand in vivo or in vitro in therapeutic orother applications where the presence of the ligand is not desired. Forexample, in cancers that are expressing large amount of bioactivezcytor19 ligand, zcytor19 soluble receptors can be used as a directantagonist of the ligand in vivo, and may aid in reducing progressionand symptoms associated with the disease. Moreover, zcytor19 solublereceptor can be used to slow the progression of cancers thatover-express zcytor19 receptors, by binding ligand in vivo that wouldotherwise enhance proliferation of those cancers. Similar in vitroapplications for a zcytor19 soluble receptor can be used, for instance,as a negative selection to select cell lines that grow in the absence ofzcytor19 ligand.

Moreover, zcytor19 soluble receptor can be used in vivo or in diagnosticapplications to detect zcytor19 ligand-expressing cancers in vivo or intissue samples. For example, the zcytor19 soluble receptor can beconjugated to a radio-label or fluorescent label as described herein,and used to detect the presence of the ligand in a tissue sample usingan in vitro ligand-receptor type binding assay, or fluorescent imagingassay. Moreover, a radiolabeled zcytor19 soluble receptor could beadministered in vivo to detect ligand-expressing solid tumors through aradio-imaging method known in the art. Similarly, zcytor19polynucleotides, polypeptides, anti-zcytor19 antibodies, or peptidebinding fragments can be used to detect zcytor19 receptor expressingcancers. In a preferred embodiment zcytor19 polynucleotides,polypeptides, anti-zcytor19 antibodies, or peptide binding fragments canbe used to detect leukemias, more preferably B-cell leukemias, and mostpreferably pre-B-cell acute lymphoblastic leukemia.

It is preferred to purify the polypeptides of the present invention to≧80% purity, more preferably to ≧90% purity, even more preferably ≧95%purity, and particularly preferred is a pharmaceutically pure state,that is greater than 99.9% pure with respect to contaminatingmacromolecules, particularly other proteins and nucleic acids, and freeof infectious and pyrogenic agents. Preferably, a purified polypeptideis substantially free of other polypeptides, particularly otherpolypeptides of animal origin.

Expressed recombinant zcytor19 polypeptides (or zcytor19 chimeric orfusion polypeptides) can be purified using fractionation and/orconventional purification methods and media. Ammonium sulfateprecipitation and acid or chaotrope extraction may be used forfractionation of samples. Exemplary purification steps may includehydroxyapatite, size exclusion, FPLC and reverse-phase high performanceliquid chromatography. Suitable chromatographic media includederivatized dextrans, agarose, cellulose, polyacrylamide, specialtysilicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred.Exemplary chromatographic media include those media derivatized withphenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia),Toyopearl butyl 650 (Toso Haas, Montgomeryville, Pa.), Octyl-Sepharose(Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG71 (Toso Haas) and the like. Suitable solid supports include glassbeads, silica-based resins, cellulosic resins, agarose beads,cross-linked agarose beads, polystyrene beads, cross-linkedpolyacrylamide resins and the like that are insoluble under theconditions in which they are to be used. These supports may be modifiedwith reactive groups that allow attachment of proteins by amino groups,carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydratemoieties. Examples of coupling chemistries include cyanogen bromideactivation, N-hydroxysuccinimide activation, epoxide activation,sulfhydryl activation, hydrazide activation, and carboxyl and aminoderivatives for carbodiimide coupling chemistries. These and other solidmedia are well known and widely used in the art, and are available fromcommercial suppliers. Methods for binding receptor polypeptides tosupport media are well known in the art. Selection of a particularmethod is a matter of routine design and is determined in part by theproperties of the chosen support. See, for example, AffinityChromatography: Principles & Methods, Pharmacia LKB Biotechnology,Uppsala, Sweden, 1988.

The polypeptides of the present invention can be isolated byexploitation of their biochemical, structural, and biologicalproperties. For example, immobilized metal ion adsorption (IMAC)chromatography can be used to purify histidine-rich proteins, includingthose comprising polyhistidine tags. Briefly, a gel is first chargedwith divalent metal ions to form a chelate (Sulkowski, Trends inBiochem. 3:1-7, 1985). Histidine-rich proteins will be adsorbed to thismatrix with differing affinities, depending upon the metal ion used, andwill be eluted by competitive elution, lowering the pH, or use of strongchelating agents. Other methods of purification include purification ofglycosylated proteins by lectin affinity chromatography and ion exchangechromatography (Methods in Enzymol., Vol. 182, “Guide to ProteinPurification”, M. Deutscher, (ed.), Acad. Press, San Diego, 1990, pp.529-39). Within additional embodiments of the invention, a fusion of thepolypeptide of interest and an affinity tag (e.g., maltose-bindingprotein, an immunoglobulin domain) may be constructed to facilitatepurification.

Moreover, using methods described in the art, polypeptide fusions, orhybrid zcytor19 proteins, are constructed using regions or domains ofthe inventive zcytor19 in combination with those of other human cytokinereceptor family proteins, or heterologous proteins (Sambrook et al.,ibid., Altschul et al., ibid., Picard, Cur. Opin. Biology, 5:511-5,1994, and references therein). These methods allow the determination ofthe biological importance of larger domains or regions in a polypeptideof interest. Such hybrids may alter reaction kinetics, binding,constrict or expand the substrate specificity, or alter tissue andcellular localization of a polypeptide, and can be applied topolypeptides of unknown structure.

Fusion polypeptides or proteins can be prepared by methods known tothose skilled in the art by preparing each component of the fusionprotein and chemically conjugating them. Alternatively, a polynucleotideencoding one or more components of the fusion protein in the properreading frame can be generated using known techniques and expressed bythe methods described herein. For example, part or all of a domain(s)conferring a biological function may be swapped between zcytor19 of thepresent invention with the functionally equivalent domain(s) fromanother cytokine family member. Such domains include, but are notlimited to, the secretory signal sequence, extracellular cytokinebinding domain, cytokine binding fragment, fibronectin type III domains,transmembrane domain, and intracellular signaling domain, as disclosedherein. Such fusion proteins would be expected to have a biologicalfunctional profile that is the same or similar to polypeptides of thepresent invention or other known family proteins, depending on thefusion constructed. Moreover, such fusion proteins may exhibit otherproperties as disclosed herein.

Standard molecular biological and cloning techniques can be used to swapthe equivalent domains between the zcytor19 polypeptide and thosepolypeptides to which they are fused. Generally, a DNA segment thatencodes a domain of interest, e.g., a zcytor19 domain described herein,is operably linked in frame to at least one other DNA segment encodingan additional polypeptide (for instance a domain or region from anothercytokine receptor, such as, interferon-gamma, alpha and beta chains andthe interferon-alpha/beta receptor alpha and beta chains, zcytor11(commonly owned U.S. Pat. No. 5,965,704), CRF2-4, DIRS1, zcytor7(commonly owned U.S. Pat. No. 5,945,511), or other class II cytokinereceptor), and inserted into an appropriate expression vector, asdescribed herein. Generally DNA constructs are made such that theseveral DNA segments that encode the corresponding regions of apolypeptide are operably linked in frame to make a single construct thatencodes the entire fusion protein, or a functional portion thereof. Forexample, a DNA construct would encode from N-terminus to C-terminus afusion protein comprising a signal polypeptide followed by a cytokinebinding domain, followed by a transmembrane domain, followed by anintracellular signaling domain. Such fusion proteins can be expressed,isolated, and assayed for activity as described herein. Moreover, suchfusion proteins can be used to express and secrete fragments of thezcytor19 polypeptide, to be used, for example to inoculate an animal togenerate anti-zcytor19 antibodies as described herein. For example asecretory signal sequence can be operably linked to extracellularcytokine binding domain, cytokine binding fragment, individualfibronectin type III domains, transmembrane domain, and intracellularsignaling domain, as disclosed herein, or a combination thereof (e.g.,operably linked polypeptides comprising a fibronectin III domainattached to a linker, or zcytor19 polypeptide fragments describedherein), to secrete a fragment of zcytor19 polypeptide that can bepurified as described herein and serve as an antigen to be inoculatedinto an animal to produce anti-zcytor19 antibodies, as described herein.

An in vivo approach for assaying proteins of the present inventioninvolves viral delivery systems. Exemplary viruses for this purposeinclude adenovirus, herpesvirus, retroviruses, vaccinia virus, andadeno-associated virus (AAV). Adenovirus, a double-stranded DNA virus,is currently the best studied gene transfer vector for delivery ofheterologous nucleic acid (for review, see T. C. Becker et al., Meth.Cell Biol. 43:161-89, 1994; and J. T. Douglas and D. T. Curiel, Science& Medicine 4:44-53, 1997). The adenovirus system offers severaladvantages: (i) adenovirus can accommodate relatively large DNA inserts;(ii) can be grown to high-titer; (iii) infect a broad range of mammaliancell types; and (iv) can be used with a large number of differentpromoters including ubiquitous, tissue specific, and regulatablepromoters. Also, because adenoviruses are stable in the bloodstream,they can be administered by intravenous injection.

In view of the tissue distribution observed for zcytor19, agonists(including the natural ligand/substrate/cofactor/etc.) and antagonistshave enormous potential in both in vitro and in vivo applications.Compounds identified as zcytor19 agonists are useful for stimulatinggrowth of immune and hematopoietic cells in vitro and in vivo. Forexample, zcytor19 soluble receptors, and agonist compounds are useful ascomponents of defined cell culture media, and may be used alone or incombination with other cytokines and hormones to replace serum that iscommonly used in cell culture. Agonists are thus useful in specificallypromoting the growth and/or development of T-cells, B-cells, and othercells of the lymphoid and myeloid lineages in culture. Moreover,zcytor19 soluble receptor, agonist, or antagonist may be used in vitroin an assay to measure stimulation of colony formation from isolatedprimary bone marrow cultures. Such assays are well known in the art.

Antagonists are also useful as research reagents for characterizingsites of ligand-receptor interaction. Inhibitors of zcytor19 activity(zcytor19 antagonists) include anti-zcytor19 antibodies and solublezcytor19 receptors, as well as other peptidic and non-peptidic agents(including ribozymes).

Zcytor19 can also be used to identify modulators (e.g, antagonists) ofits activity. Test compounds are added to the assays disclosed herein toidentify compounds that inhibit the activity of zcytor19. In addition tothose assays disclosed herein, samples can be tested for inhibition ofzcytor19 activity within a variety of assays designed to measurezcytor19 binding, oligomerization, or the stimulation/inhibition ofzcytor19-dependent cellular responses.

A zcytor19 ligand-binding polypeptide, such as the extracellular domainor cytokine binding domain disclosed herein, can also be used forpurification of ligand. The polypeptide is immobilized on a solidsupport, such as beads of agarose, cross-linked agarose, glass,cellulosic resins, silica-based resins, polystyrene, cross-linkedpolyacrylamide, or like materials that are stable under the conditionsof use. Methods for linking polypeptides to solid supports are known inthe art, and include amine chemistry, cyanogen bromide activation,N-hydroxysuccinimide activation, epoxide activation, sulthydrylactivation, and hydrazide activation. The resulting medium willgenerally be configured in the form of a column, and fluids containingligand are passed through the column one or more times to allow ligandto bind to the receptor polypeptide. The ligand is then eluted usingchanges in salt concentration, chaotropic agents (guanidine HCl), or pHto disrupt ligand-receptor binding.

An assay system that uses a ligand-binding receptor (or an antibody, onemember of a complement/anti-complement pair) or a binding fragmentthereof, and a commercially available biosensor instrument may beadvantageously employed (e.g., BIAcore™, Pharmacia Biosensor,Piscataway, N.J.; or SELDI™ technology, Ciphergen, Inc., Palo Alto,Calif.). Such receptor, antibody, member of a complement/anti-complementpair or fragment is immobilized onto the surface of a receptor chip. Useof this instrument is disclosed by Karlsson, J. Immunol. Methods145:229-240, 1991 and Cunningham and Wells, J. Mal. Biol. 234:554-63,1993.

Ligand-binding receptor polypeptides can also be used within other assaysystems known in the art. Such systems include Scatchard analysis fordetermination of binding affinity (see Scatchard, Ann. NY Acad. Sci. 51:660-672, 1949) and calorimetric assays (Cunningham et al., Science253:545-48, 1991; Cunningham et al., Science 245:821-25, 1991).

Zcytor19 polypeptides can also be used to prepare antibodies that bindto zcytor19 epitopes, peptides or polypeptides. The zcytor19 polypeptideor a fragment thereof serves as an antigen (immunogen) to inoculate ananimal and elicit an immune response. One of skill in the art wouldrecognize that antigenic, epitope-bearing polypeptides contain asequence of at least 6, preferably at least 9, and more preferably atleast 15 to about 30 contiguous amino acid residues of a zcytor19polypeptide (e.g., SEQ ID NO:2, SEQ ID NO:19 or SEQ ID NO:21).Polypeptides comprising a larger portion of a zcytor19 polypeptide,i.e., from 30 to 100 residues up to the entire length of the amino acidsequence are included. Antigens or immunogenic epitopes can also includeattached tags, adjuvants and carriers, as described herein. Suitableantigens include the zcytor19 polypeptide encoded by SEQ ID NO:2 fromamino acid number 21 (Arg) to amino acid number 491 (Arg), or acontiguous 9 to 471 amino acid fragment thereof. Suitable antigens alsoinclude the zcytor19 polypeptide encoded by SEQ ID NO:19 from amino acidnumber 21 (Arg) to amino acid number 520 (Arg), or a contiguous 9 to 500amino acid fragment thereof; and the truncated soluble zcytor19polypeptide encoded by SEQ ID NO:21 from amino acid number 21 (Arg) toamino acid number 211 (Ser), or a contiguous 9 to 191 amino acidfragment thereof. Preferred peptides to use as antigens are theextracellular cytokine binding domain, cytokine binding fragment,fibronectin type III domains, intracellular signaling domain, or otherdomains and motifs disclosed herein, or a combination thereof; andzcytor19 hydrophilic peptides such as those predicted by one of skill inthe art from a hydrophobicity plot, determined for example, from aHopp/Woods hydrophilicity profile. Zcytor19 hydrophilic peptides includepeptides comprising amino acid sequences selected from the groupconsisting of: (1) residues 295 through 300 of SEQ ID NO:2; (2) residues451 through 456 of SEQ ID NO:2; (3) residues 301 through 306 of SEQ IDNO:2; (4) residues 294 through 299 of SEQ ID NO:2; and (5) residues 65through 70 of SEQ ID NO:2. In addition, zcytor19 antigenic epitopes aspredicted by a Jameson-Wolf plot, e.g., using DNASTAR Protean program(DNASTAR, Inc., Madison, Wis.) are suitable antigens. In addition,conserved motifs, and variable regions between conserved motifs ofzcytor19 are suitable antigens. Antibodies generated from this immuneresponse can be isolated and purified as described herein. Methods forpreparing and isolating polyclonal and monoclonal antibodies are wellknown in the art. See, for example, Current Protocols in Immunology,Cooligan, et al. (eds.), National Institutes of Health, John Wiley andSons, Inc., 1995; Sambrook et al., Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor, N.Y., 1989; and Hurrell, J.G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques andApplications, CRC Press, Inc., Boca Raton, Fla., 1982.

As would be evident to one of ordinary skill in the art, polyclonalantibodies can be generated from inoculating a variety of warm-bloodedanimals such as horses, cows, goats, sheep, dogs, chickens, rabbits,mice, and rats with a zcytor19 polypeptide or a fragment thereof. Theimmunogenicity of a zcytor19 polypeptide may be increased through theuse of an adjuvant, such as alum (aluminum hydroxide) or Freund'scomplete or incomplete adjuvant. Polypeptides useful for immunizationalso include fusion polypeptides, such as fusions of zcytor19 or aportion thereof with an immunoglobulin polypeptide or with maltosebinding protein. The polypeptide immunogen may be a full-length moleculeor a portion thereof. If the polypeptide portion is “hapten-like”, suchportion may be advantageously joined or linked to a macromolecularcarrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin(BSA) or tetanus toxoid) for immunization.

As used herein, the term “antibodies” includes polyclonal antibodies,affinity-purified polyclonal antibodies, monoclonal antibodies, andantigen-binding fragments, such as F(ab′)₂ and Fab proteolyticfragments. Genetically engineered intact antibodies or fragments, suchas chimeric antibodies, Fv fragments, single chain antibodies and thelike, as well as synthetic antigen-binding peptides and polypeptides,are also included. Non-human antibodies may be humanized by graftingnon-human CDRs onto human framework and constant regions, or byincorporating the entire non-human variable domains (optionally“cloaking” them with a human-like surface by replacement of exposedresidues, wherein the result is a “veneered” antibody). In someinstances, humanized antibodies may retain non-human residues within thehuman variable region framework domains to enhance proper bindingcharacteristics. Through humanizing antibodies, biological half-life maybe increased, and the potential for adverse immune reactions uponadministration to humans is reduced. Moreover, human antibodies can beproduced in transgenic, non-human animals that have been engineered tocontain human immunoglobulin genes as disclosed in WIPO Publication WO98/24893. It is preferred that the endogenous immunoglobulin genes inthese animals be inactivated or eliminated, such as by homologousrecombination. Antibodies in the present invention include, but are notlimited to, antibodies that bind the zcytor19/CRF2-4 heterodimer, aswell as the heterodimeric soluble receptor complex.

Alternative techniques for generating or selecting antibodies usefulherein include in vitro exposure of lymphocytes to zcytor19 protein orpeptide, and selection of antibody display libraries in phage or similarvectors (for instance, through use of immobilized or labeled zcytor19protein or peptide). Techniques for creating and screening such randompeptide display libraries are known in the art (Ladner et al., U.S. Pat.No. 5,223,409; Ladner et al., U.S. Pat. No. 4,946,778; Ladner et al.,U.S. Pat. No. 5,403,484 and Ladner et al., U.S. Pat. No. 5,571,698) andrandom peptide display libraries and kits for screening such librariesare available commercially, for instance from Clontech (Palo Alto,Calif.), Invitrogen Inc. (San Diego, Calif.), New England Biolabs, Inc.(Beverly, Mass.) and Pharmacia LKB Biotechnology Inc. (Piscataway,N.J.). Random peptide display libraries can be screened using thezcytor19 sequences disclosed herein to identify proteins which bind tozcytor19. These “binding peptides” which interact with zcytor19polypeptides can be used for tagging cells, e.g., such as those in whichzcytor19 is specifically expressed; for isolating homolog polypeptidesby affinity purification; they can be directly or indirectly conjugatedto drugs, toxins, radionuclides and the like. These binding peptides canalso be used in analytical methods such as for screening expressionlibraries and neutralizing activity. The binding peptides can also beused for diagnostic assays for determining circulating levels ofzcytor19 polypeptides; for detecting or quantitating soluble zcytor19polypeptides as marker of underlying pathology or disease. These bindingpeptides can also act as zcytor19 “antagonists” to block zcytor19binding and signal transduction in vitro and in vivo. Theseanti-zcytor19 binding peptides would be useful for inhibiting the actionof a ligand that binds with zcytor19.

Antibodies are considered to be specifically binding if: 1) they exhibita threshold level of binding activity, and 2) they do not significantlycross-react with related polypeptide molecules. A threshold level ofbinding is determined if anti-zcytor19 antibodies herein bind to azcytor19 polypeptide, peptide or epitope with an affinity at least10-fold greater than the binding affinity to control (non-zcytor19)polypeptide. It is preferred that the antibodies exhibit a bindingaffinity (K_(a)) of 10⁶ M⁻¹ or greater, preferably 10⁷ M⁻¹ or greater,more preferably 10⁸ M⁻¹ or greater, and most preferably 10⁹ M⁻¹ orgreater. The binding affinity of an antibody can be readily determinedby one of ordinary skill in the art, for example, by Scatchard analysis(Scatchard, G., Ann. NY Acad. Sci. 51: 660-672, 1949).

Whether anti-zcytor19 antibodies do not significantly cross-react withrelated polypeptide molecules is shown, for example, by the antibodydetecting zcytor19 polypeptide but not known related polypeptides usinga standard Western blot analysis (Ausubel et al., ibid.). Examples ofknown related polypeptides are those disclosed in the prior art, such asknown orthologs, and paralogs, and similar known members of a proteinfamily (e.g., class II cytokine receptors, for example,interferon-gamma, alpha and beta chains and the interferon-alpha/betareceptor alpha and beta chains, zcytor11 (commonly owned U.S. Pat. No.5,965,704), CRF2-4, DIRS1, zcytor7 (commonly owned U.S. Pat. No.5,945,511) receptors). Screening can also be done using non-humanzcytor19, and zcytor19 mutant polypeptides. Moreover, using routinemethods, antibodies can be “screened against” known relatedpolypeptides, to isolate a population that specifically binds to thezcytor19 polypeptides. Screening allows isolation of polyclonal andmonoclonal antibodies non-crossreactive to known closely relatedpolypeptides (Antibodies: A Laboratory Manual, Harlow and Lane (eds.),Cold Spring Harbor Laboratory Press, 1988; Current Protocols inImmunology, Cooligan, et al. (eds.), National Institutes of Health, JohnWiley and Sons, Inc., 1995). Screening and isolation of specificantibodies is well known in the art. See, Fundamental Immunology, Paul(eds.), Raven Press, 1993; Getzoff et al., Adv. in Immunol. 43: 1-98,1988; Monoclonal Antibodies: Principles and Practice, Goding, J. W.(eds.), Academic Press Ltd., 1996; Benjamin et al., Ann. Rev. Immunol.2: 67-101, 1984. Specifically binding anti-zcytor19 antibodies can bedetected by a number of methods in the art, and disclosed below.

A variety of assays known to those skilled in the art can be utilized todetect antibodies which specifically bind to zcytor19 proteins orpeptides. Exemplary assays are described in detail in Antibodies: ALaboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor LaboratoryPress, 1988. Representative examples of such assays include: concurrentimmunoelectrophoresis, radioimmunoassay, radioimmuno-precipitation,enzyme-linked immunosorbent assay (ELISA), dot blot or Western blotassay, inhibition or competition assay, and sandwich assay. In addition,antibodies can be screened for binding to wild-type versus mutantzcytor19 protein or polypeptide.

Antibodies to zcytor19 may be used for tagging cells that expresszcytor19; for isolating zcytor19 by affinity purification; fordiagnostic assays for determining circulating levels of zcytor19polypeptides; for detecting or quantitating soluble zcytor19 as markerof underlying pathology or disease; for detecting or quantitating in ahistologic, biopsy, or tissue sample zcytor19 receptor as marker ofunderlying pathology or disease; for stimulating cytotoxicity or ADCC onzcytor19-bearing cancer cells; in analytical methods employing FACS; forscreening expression libraries; for generating anti-idiotypicantibodies; and as neutralizing antibodies or as antagonists to blockzcytor19 activity in vitro and in vivo. Antibodies herein may also bedirectly or indirectly conjugated to drugs, toxins, radionuclides andthe like, and these conjugates used for in vivo diagnostic ortherapeutic applications. Moreover, antibodies to zcytor19 or fragmentsthereof may be used in vitro to detect denatured zcytor19 or fragmentsthereof in assays, for example, Western Blots or other assays known inthe art.

Antibodies herein can also be directly or indirectly conjugated todrugs, toxins, radionuclides and the like, and these conjugates used forin vivo diagnostic or therapeutic applications.

Suitable detectable molecules may be directly or indirectly attached topolypeptides that bind zcytor19 (“binding polypeptides,” includingbinding peptides disclosed above), antibodies, or bioactive fragments orportions thereof. Suitable detectable molecules include radionuclides,enzymes, substrates, cofactors, inhibitors, fluorescent markers,chemiluminescent markers, magnetic particles and the like. Suitablecytotoxic molecules may be directly or indirectly attached to thepolypeptide or antibody, and include bacterial or plant toxins (forinstance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin and thelike), as well as therapeutic radionuclides, such as iodine-131,rhenium-188 or yttrium-90 (either directly attached to the polypeptideor antibody, or indirectly attached through means of a chelating moiety,for instance). Binding polypeptides or antibodies may also be conjugatedto cytotoxic drugs, such as adriamycin. For indirect attachment of adetectable or cytotoxic molecule, the detectable or cytotoxic moleculecan be conjugated with a member of a complementary/anticomplementarypair, where the other member is bound to the binding polypeptide orantibody portion. For these purposes, biotin/streptavidin is anexemplary complementary/anticomplementary pair.

In another embodiment, binding polypeptide-toxin fusion proteins orantibody-toxin fusion proteins can be used for targeted cell or tissueinhibition or ablation (for instance, to treat cancer cells or tissues,e.g., such as those specific tissues and tumors wherein zcytor19 isexpressed). Alternatively, if the binding polypeptide has multiplefunctional domains (i.e., an activation domain or a ligand bindingdomain, plus a targeting domain), a fusion protein including only thetargeting domain may be suitable for directing a detectable molecule, acytotoxic molecule or a complementary molecule to a cell or tissue typeof interest. In instances where the fusion protein including only asingle domain includes a complementary molecule, the anti-complementarymolecule can be conjugated to a detectable or cytotoxic molecule. Suchdomain-complementary molecule fusion proteins thus represent a generictargeting vehicle for cell/tissue-specific delivery of genericanti-complementary-detectable/cytotoxic molecule conjugates. Similarly,in another embodiment, zcytor19 binding polypeptide-cytokine orantibody-cytokine fusion proteins can be used for enhancing in vivokilling of target tissues, if the binding polypeptide-cytokine oranti-zcytor19 antibody targets the hyperproliferative cell (See,generally, Hornick et al., Blood 89:4437-47, 1997). They describedfusion proteins enable targeting of a cytokine to a desired site ofaction, thereby providing an elevated local concentration of cytokine.Suitable anti-zcytor19 antibodies target an undesirable cell or tissue(i.e., a tumor or a leukemia), and the fused cytokine mediates improvedtarget cell lysis by effector cells. Suitable cytokines for this purposeinclude interleukin 2 and granulocyte-macrophage colony-stimulatingfactor (GM-CSF), for instance.

Alternatively, zcytor19 binding polypeptide or antibody fusion proteinsdescribed herein can be used for enhancing in vivo killing of targettissues by directly stimulating a zcytor19-modulated apoptotic pathway,resulting in cell death of hyperproliferative cells expressing zcytor19.

The bioactive binding polypeptide or antibody conjugates describedherein can be delivered orally, intravenously, intraarterially orintraductally, or may be introduced locally at the intended site ofaction.

Moreover, anti-zcytor19 antibodies and binding fragments can be used fortagging and sorting cells that specifically-express Zcytor19, such asbone marrow and thyroid cells, and other cells, described herein. Suchmethods of cell tagging and sorting are well known in the art (see,e.g., “Molecular Biology of the Cell”, 3^(rd) Ed., Albert, B. et al.(Garland Publishing, London & New York, 1994). One of skill in the artwould recognize the importance of separating cell tissue types to studycells, and the use of antibodies to separate specific cell tissue types.

Antisense methodology can be used to inhibit zcytor19 genetranscription, such as to inhibit cell proliferation in vivo.Polynucleotides that are complementary to a segment of azcytor19-encoding polynucleotide (e.g., a polynucleotide as set forth inSEQ ID NO:1 SEQ ID NO:18, or SEQ ID NO:20) are designed to bind tozcytor19-encoding mRNA and to inhibit translation of such mRNA. Suchantisense polynucleotides are used to inhibit expression of zcytor19polypeptide-encoding genes in cell culture or in a subject.

In addition, as a cell surface molecule, zcytor19 polypeptides can beused as a target to introduce gene therapy into a cell. This applicationwould be particularly appropriate for introducing therapeutic genes intocells in which zcytor19 is normally expressed, such as lymphoid tissue,bone marrow, prostate, thyroid, and PBLs, or cancer cells which expresszcytor19 polypeptide. For example, viral gene therapy, such as describedabove, can be targeted to specific cell types in which express acellular receptor, such as zcytor19 polypeptide, rather than the viralreceptor. Antibodies, or other molecules that recognize zcytor19molecules on the target cell's surface can be used to direct the virusto infect and administer gene therapeutic material to that target cell.See, Woo, S. L. C, Nature Biotech. 14:1538, 1996; Wickham, T. J. et al,Nature Biotech. 14:1570-1573, 1996; Douglas, J. T et al., NatureBiotech. 14:1574-1578, 1996; Rihova, B., Crit. Rev. Biotechnol.17:149-169, 1997; and Vile, R. G. et al., Mol. Med. Today 4:84-92, 1998.For example, a bispecific antibody containing a virus-neutralizing Fabfragment coupled to a zcytor19-specific antibody can be used to directthe virus to cells expressing the zcytor19 receptor and allow efficiententry of the virus containing a genetic element into the cells. See, forexample, Wickham, T. J., et al., J. Virol. 71:7663-7669, 1997; andWickham, T. J., et al., J. Virol. 70:6831-6838, 1996.

The present invention also provides reagents which will find use indiagnostic applications. For example, the zcytor19 gene, a probecomprising zcytor19 DNA or RNA or a subsequence thereof can be used todetermine if the zcytor19 gene is present on chromosome 1 or if amutation has occurred. Zcytor19 is located at the 1p36.11 region ofchromosome 1. Detectable chromosomal aberrations at the zcytor19 genelocus include, but are not limited to, aneuploidy, gene copy numberchanges, insertions, deletions, restriction site changes andrearrangements. Such aberrations can be detected using polynucleotidesof the present invention by employing molecular genetic techniques, suchas restriction fragment length polymorphism (RFLP) analysis,fluorescence in situ hybridization methods, short tandem repeat (STR)analysis employing PCR techniques, and other genetic linkage analysistechniques known in the art (Sambrook et al., ibid.; Ausubel et. al.,ibid.; Marian, Chest 108:255-65, 1995).

The precise knowledge of a gene's position can be useful for a number ofpurposes, including: 1) determining if a sequence is part of an existingcontig and obtaining additional surrounding genetic sequences in variousforms, such as YACs, BACs or cDNA clones; 2) providing a possiblecandidate gene for an inheritable disease which shows linkage to thesame chromosomal region; and 3) cross-referencing model organisms, suchas mouse, which may aid in determining what function a particular genemight have.

The zcytor19 gene is located at the 1p36.11 region of chromosome 1. Oneof skill in the art would recognize that chromosomal aberrations in andaround the 1p36 region are involved in several cancers includingneuroblastoma, melanoma, breast, colon, prostate and other cancers. Suchaberrations include gross chromosomal abnormalities such astranslocations, loss of heterogeneity (LOH) and the like in and around1p36. Thus, a marker in the 1p36.11 locus, such as provided by thepolynucleotides of the present invention, would be useful in detectingtranslocations, aneuploidy, rearrangements, LOH other chromosomalabnormalities involving this chromosomal region that are present incancers. For example, zcytor19 polynucleotide probes can be used todetect abnormalities or genotypes associated with neuroblastoma, whereinLOH between 1p36.1 and 1p36.3 is prevalent, and a breakpoint at 1p36.1is evident. At least 70% of neuroblastomas have cytogenetically visiblechromosomal aberrations in 1p, including translocation and deletion, andthat the abnormality is most likely due to complex translocation anddeletion mechanisms. See, for example Ritke, M K et al., Cytogenet. CellGenet. 50:84-90, 1989; and Weith, A et al., Genes Chromosomes Cancer1:159-166, 1989). As zcytor19 is localized to 1p36.11, and fallsdirectly within the region wherein aberrations are prevalent inneuroblastoma, one of skill in the art would appreciate that thepolynucleotides of the present invention could serve as a diagnostic forneuroblastoma, as well as aid in the elucidation of translocation anddeletion mechanisms that give rise to neuroblastoma. In addition, LOH at1p36 is evident in melanoma (Dracopoli, N C et al, Am. J. Hum. Genet. 45(suppl.):A19, 1989; Dracopoli, N C et al, Proc. Nat. Acad. Sci.86:4614-4618, 1989; Goldstein, A M et al., Am. J. Hum. Genet.52:537-550, 1993); as well as prostate cancer in families with a historyof both prostate and brain cancer (1p36, LOH) (Gibbs, M et al., Am. J.Hum. Genet. 64:776-787, 1999); and breast cancer, wherein deletions andduplications of chromosome 1 are the most common aberrations in breastcarcinoma (1p36) (Kovacs, G. Int. J. Cancer 21:688-694, 1978; Rodgers, Cet al., Cancer Genet. Cytogent. 13:95-119, 1984; and Genuardi, M. etal., Am. J. Hum. Genet. 45:73-82, 1989). Since translocation, LOH andother aberrations in this region of human chromosome 1 are so prevalentin human cancers, and the zcytor19 gene is specifically localized to1p36.11, the polynucleotides of the present invention have use indetecting such aberrations that are clearly associated with humandisease, as described herein.

Moreover, there is further evidence for cancer resulting from mutationsin the 1p36 region wherein zcytor19 is located, and polynucleotideprobes can be used to detect abnormalities or genotypes associatedtherewith: P73, a potential tumor suppressor maps to 1p36 a regionfrequently deleted in neuroblastoma and other cancers (Kaghad, M et al.,Cell 90:809-819, 1997); rhabdomyosarcoma, which involves a translocationat the 1p36.2-p36.12 region of chromosome 1 that results in a fusion ofthe PAX7 gene from chromosome 1 with FKHR gene on chromosome 13;Leukemia-associated Protein (LAP) (1p36.1-p35) is increased in the cellsof various types of leukemia; heparin sulfate proteoglycan (Perlecan)(1p36.1) associated with tumors, and wherein translocations are seen;and colon cancer (1p36-p35). Further, zcytor19 polynucleotide probes canbe used to detect abnormalities or genotypes associated with chromosome1p36.11 deletions and translocations associated with human diseases, andpreferably cancers, as described above. Moreover, amongst other geneticloci, those for C1q complement components (C1QA, B, and G)(1p36.3-p34.1); dyslexia (1p36-p34); lymphoid activation antigen CD30(1p36); sodium channel non-voltage-gated type 1 (1p36.3-p36.2); tumornecrosis factor receptors (TNFRSF1b and INFRS12) (1p36.3-p36.2) whichlike zcytor19 are cytokine receptors; phospholipase A2 (PLA2) (1p35);rigid spine muscular dystrophy (1p36-p35) all manifest themselves inhuman disease states as well as map to this region of the human genome.See the Online Mendellian Inheritance of Man (OMIM™, National Center forBiotechnology Information, National Library of Medicine. Bethesda, Md.)gene map, and references therein, for this region of human chromosome 1on a publicly available world wide web server(http://www3.ncbi.nlm.nih.gov/htbin-post/Omim/getmap?chromosome=1p36).All of these serve as possible candidate genes for an inheritabledisease which show linkage to the same chromosomal region as thezcytor19 gene. Thus, zcytor19 polynucleotide probes can be used todetect abnormalities or genotypes associated with these defects.

Similarly, defects in the zcytor19 gene itself may result in a heritablehuman disease state. The zcytor19 gene (1p36.11) is located near anotherclass II receptor, the zcytor11 cytokine receptor gene (1p35.1)(commonly owned U.S. Pat. No. 5,965,704), as well as TNF receptors(1p36.3-p36.2), suggesting that this chromosomal region is commonlyregulated, and/or important for immune function. Moreover, one of skillin the art would appreciate that defects in cytokine receptors are knownto cause disease states in humans. For example, growth hormone receptormutation results in dwarfism (Amselem, S et al., New Eng. J. Med. 321:989-995, 1989), IL-2 receptor gamma mutation results in severe combinedimmunodeficiency (SCID) (Noguchi, M et al., Cell 73: 147-157, 1993),c-Mp1 mutation results in thrombocytopenia (Ihara, K et al., Proc. Nat.Acad. Sci. 96: 3132-3136, 1999), and severe mycobacterial and Salmonellainfections result in interleukin-12 receptor-deficient patients (deJong, R et al., Science 280: 1435-1438, 1998), amongst others. Thus,similarly, defects in zcytor19 can cause a disease state orsusceptibility to disease or infection. As, zcytor19 is a cytokinereceptor in a chromosomal hot spot for aberrations involved in numerouscancers and is shown to be expressed in pre-B-cell acute leukemia cells,and other cancers described herein, the molecules of the presentinvention could also be directly involved in cancer formation ormetastasis. As the zcytor19 gene is located at the 1p36.11 regionzcytor19, polynucleotide probes can be used to detect chromosome 1p36.11loss, trisomy, duplication or translocation associated with humandiseases, such as immune cell cancers, neuroblastoma, bone marrowcancers, thyroid, parathyroid, prostate, melanoma, or other cancers, orimmune diseases. Moreover, molecules of the present invention, such asthe polypeptides, antagonists, agonists, polynucleotides and antibodiesof the present invention would aid in the detection, diagnosisprevention, and treatment associated with a zcytor19 genetic defect.

Mutations associated with the zcytor19 locus can be detected usingnucleic acid molecules of the present invention by employing standardmethods for direct mutation analysis, such as restriction fragmentlength polymorphism analysis, short tandem repeat analysis employing PCRtechniques, amplification-refractory mutation system analysis,single-strand conformation polymorphism detection, RNase cleavagemethods, denaturing gradient gel electrophoresis, fluorescence-assistedmismatch analysis, and other genetic analysis techniques known in theart (see, for example, Mathew (ed.), Protocols in Human MolecularGenetics (Humana Press, Inc. 1991), Marian, Chest 108:255 (1995),Coleman and Tsongalis, Molecular Diagnostics (Human Press, Inc. 1996),Elles (ed.) Molecular Diagnosis of Genetic Diseases (Humana Press, Inc.1996), Landegren (ed.), Laboratory Protocols for Mutation Detection(Oxford University Press 1996), Birren et al. (eds.), Genome Analysis,Vol. 2: Detecting Genes (Cold Spring Harbor Laboratory Press 1998),Dracopoli et al. (eds.), Current Protocols in Human Genetics (John Wiley& Sons 1998), and Richards and Ward, “Molecular Diagnostic Testing,” inPrinciples of Molecular Medicine, pages 83-88 (Humana Press, Inc.1998)). Direct analysis of an zcytor19 gene for a mutation can beperformed using a subject's genomic DNA. Methods for amplifying genomicDNA, obtained for example from peripheral blood lymphocytes, arewell-known to those of skill in the art (see, for example, Dracopoli etal. (eds.), Current Protocols in Human Genetics, at pages 7.1.6 to 7.1.7(John Wiley & Sons 1998)).

Mice engineered to express the zcytor19 gene, referred to as “transgenicmice,” and mice that exhibit a complete absence of zcytor19 genefunction, referred to as “knockout mice,” may also be generated(Snouwaert et al., Science 257:1083, 1992; Lowell et al., Nature366:740-42, 1993; Capecchi, M. R., Science 244: 1288-1292, 1989;Palmiter, R. D. et al. Annu Rev Genet. 20: 465-499, 1986). For example,transgenic mice that over-express zcytor19, either ubiquitously or undera tissue-specific or tissue-restricted promoter can be used to askwhether over-expression causes a phenotype. For example, over-expressionof a wild-type zcytor19 polypeptide, polypeptide fragment or a mutantthereof may alter normal cellular processes, resulting in a phenotypethat identifies a tissue in which zcytor19 expression is functionallyrelevant and may indicate a therapeutic target for the zcytor19, itsagonists or antagonists. For example, a preferred transgenic mouse toengineer is one that expresses a “dominant-negative” phenotype, such asone that over-expresses the zcytor19 polypeptide comprising anextracellular cytokine binding domain with the transmembrane domainattached (approximately amino acids 21 (Arg) to 249 (Trp) of SEQ ID NO:2or SEQ ID NO:19; or SEQ ID NO:4 attached in frame to a transmembranedomain). Another preferred transgenic mouse is one that over-expresseszcytor19 soluble receptors, such as those disclosed herein. Moreover,such over-expression may result in a phenotype that shows similaritywith human diseases. Similarly, knockout zcytor19 mice can be used todetermine where zcytor19 is absolutely required in vivo. The phenotypeof knockout mice is predictive of the in vivo effects of a zcytor19antagonist, such as those described herein, may have. The mouse or thehuman zcytor19 cDNA can be used to isolate murine zcytor19 mRNA, cDNAand genomic DNA, which are subsequently used to generate knockout mice.These transgenic and knockout mice may be employed to study the zcytor19gene and the protein encoded thereby in an in vivo system, and can beused as in vivo models for corresponding human or animal diseases (suchas those in commercially viable animal populations). The mouse models ofthe present invention are particularly relevant as tumor models for thestudy of cancer biology and progression. Such models are useful in thedevelopment and efficacy of therapeutic molecules used in human cancers.Because increases in zcytor19 expression, as well as decreases inzcytor19 expression are associated with specific human cancers, bothtransgenic mice and knockout mice would serve as useful animal modelsfor cancer. Moreover, in a preferred embodiment, zcytor19 transgenicmouse can serve as an animal model for specific tumors, particularlyesophagus, liver, ovary, rectum, stomach, and uterus tumors, andmelanoma, B-cell leukemia and other lymphoid cancers. Moreover,transgenic mice expression of zcytor19 antisense polynucleotides orribozymes directed against zcytor19, described herein, can be usedanalogously to transgenic mice described above.

For pharmaceutical use, the soluble receptor polypeptides of the presentinvention are formulated for parenteral, particularly intravenous orsubcutaneous, delivery according to conventional methods. Intravenousadministration will be by bolus injection or infusion over a typicalperiod of one to several hours. In general, pharmaceutical formulationswill include a zcytor19 soluble receptor polypeptide in combination witha pharmaceutically acceptable vehicle, such as saline, buffered saline,5% dextrose in water or the like. Formulations may further include oneor more excipients, preservatives, solubilizers, buffering agents,albumin to prevent protein loss on vial surfaces, etc. Methods offormulation are well known in the art and are disclosed, for example, inRemington: The Science and Practice of Pharmacy, Gennaro, ed., MackPublishing Co., Easton, Pa., 19th ed., 1995. Therapeutic doses willgenerally be in the range of 0.1 to 100 μg/kg of patient weight per day,preferably 0.5-20 mg/kg per day, with the exact dose determined by theclinician according to accepted standards, taking into account thenature and severity of the condition to be treated, patient traits, etc.Determination of dose is within the level of ordinary skill in the art.The proteins may be administered for acute treatment, over one week orless, often over a period of one to three days or may be used in chronictreatment, over several months or years. In general, a therapeuticallyeffective amount of zcytor19 soluble receptor polypeptide is an amountsufficient to produce a clinically significant effect.

Polynucleotides and polypeptides of the present invention willadditionally find use as educational tools as a laboratory practicumkits for courses related to genetics and molecular biology, proteinchemistry and antibody production and analysis. Due to its uniquepolynucleotide and polypeptide sequence molecules of zcytor19 can beused as standards or as “unknowns” for testing purposes. For example,zcytor19 polynucleotides can be used as an aid, such as, for example, toteach a student how to prepare expression constructs for bacterial,viral, and/or mammalian expression, including fusion constructs, whereinzcytor19 is the gene to be expressed; for determining the restrictionendonuclease cleavage sites of the polynucleotides; determining mRNA andDNA localization of zcytor19 polynucleotides in tissues (i.e., byNorthern and Southern blotting as well as polymerase chain reaction);and for identifying related polynucleotides and polypeptides by nucleicacid hybridization.

Zcytor19 polypeptides can be used educationally as an aid to teachpreparation of antibodies; identifying proteins by Western blotting;protein purification; determining the weight of expressed zcytor19polypeptides as a ratio to total protein expressed; identifying peptidecleavage sites; coupling amino and carboxyl terminal tags; amino acidsequence analysis, as well as, but not limited to monitoring biologicalactivities of both the native and tagged protein (i.e., receptorbinding, signal transduction, proliferation, and differentiation) invitro and in vivo. Zcytor19 polypeptides can also be used to teachanalytical skills such as mass spectrometry, circular dichroism todetermine conformation, especially of the four alpha helices, x-raycrystallography to determine the three-dimensional structure in atomicdetail, nuclear magnetic resonance spectroscopy to reveal the structureof proteins in solution. For example, a kit containing the zcytor19 canbe given to the student to analyze. Since the amino acid sequence wouldbe known by the professor, the specific protein can be given to thestudent as a test to determine the skills or develop the skills of thestudent, the teacher would then know whether or not the student hascorrectly analyzed the polypeptide. Since every polypeptide is unique,the educational utility of zcytor19 would be unique unto itself.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1 Identification and Isolation of Full-Length HumanZcytor19 cDNA

Zcytor19 was identified as a predicted full-length cDNA from humangenomic DNA AL358412 (Genbank). The sequence of the predicted fulllength zcytor19 polynucleotide is shown in SEQ ID NO:1 and thecorresponding polypeptide is shown in SEQ ID NO:2. A variant full-lengthzcytor19 cDNA sequence was identified and is shown in SEQ ID NO:18 andthe corresponding polynucleotides shown in SEQ ID NO:19. Moreover, atrucnated soluble form of zcytor19 cDNA sequence was identified and isshown in SEQ ID NO:20 and the corresponding polynucleotides shown in SEQID NO:21.

Example 2 Tissue Distribution in Tissue Panels Using Northern Blot andPCR A. Human Zcytor19 Tissue Distribution Using Northern Blot

Human Multiple Tissue Northern Blots (Human 12-lane MTN Blot I and II,and Human Immune System MTN Blot II) (Clontech) are probed to determinethe tissue distribution of human zcytor19 expression. A PCR derivedprobe that hybridizes to SEQ ID NO:1 or SEQ ID NO:18 is amplified usingstandard PCR amplification methods. An exemplary PCR reaction is carriedout as follows using primers designed to hybridize to SEQ ID NO:1, SEQID NO:18 or its complement: 30 cycles of 94° C. for 1 minute, 65° C. for1 minute, and 72° C. for 1 minute; followed by 1 cycle at 72° C. for 7minutes. The PCR product is visualized by agarose gel electrophoresisand the PCR product is gel purified as described herein. The probe isradioactively labeled using, e.g., the PRIME IT II™ Random PrimerLabeling Kit (Stratagene) according to the manufacturer's instructions.The probe is purified using, e.g., a NUCTRAP™ push column (Stratagene).EXPRESSHYB™ (Clontech) solution is used for the prehybridization and asa hybridizing solution for the Northern blots. Prehybridization iscarried out, for example, at 68° C. for 2 hours. Hybridization takesplace overnight at about 68° C. with about 1.5×10⁶ cpm/ml of labeledprobe. The blots are washed three times at room temperature in 2×SSC,0.05% SDS, followed by 1 wash for 10 minutes in 2×SSC, 0.1% SDS at 50°C. After exposure to X-ray film, a transcript corresponding to thelength of SEQ ID NO:1 SEQ ID NO:18, or SEQ ID NO:20 or of an mRNAencoding SEQ ID NO:2, SEQ ID NO:19 or SEQ ID NO:21 is expected to beseen in tissues that specifically express zcytor19, but not othertissues.

Northern analysis is also performed using Human Cancer Cell Line MTN™(Clontech). PCR and probing conditions are as described above. A strongsignal in a cancer line suggests that zcytor19 expression may beexpressed in activated cells and/or may indicate a cancerous diseasestate. Moreover, using methods known in the art, Northern blots or PCRanalysis of activated lymphocyte cells can also show whether zcytor19 isexpressed in activated immune cells. Based on electronic Northerninformation zcytor19 was shown to be expressed specifically in pre-Bcell acute lymphoblastic leukemia cells.

B. Tissue Distribution in Tissue Panels Using PCR

A panel of cDNAs from human tissues was screened for zcytor19 expressionusing PCR. The panel was made in-house and contained 94 marathon cDNAand cDNA samples from various normal and cancerous human tissues andcell lines are shown in Table 5, below. The cDNAs came from in-houselibraries or marathon cDNAs from in-house RNA preps, Clontech RNA, orInvitrogen RNA. The marathon cDNAs were made using the marathon-Ready™kit (Clontech, Palo Alto, Calif.) and QC tested with clathrin primersZC21195 (SEQ ID NO:6) and ZC21196 (SEQ ID NO:7) and then diluted basedon the intensity of the clathrin band. To assure quality of the panelsamples, three tests for quality control (QC) were run: (1) To assessthe RNA quality used for the libraries, the in-house cDNAs were testedfor average insert size by PCR with vector oligos that were specific forthe vector sequences for an individual cDNA library; (2) Standardizationof the concentration of the cDNA in panel samples was achieved usingstandard PCR methods to amplify full length alpha tubulin or G3PDH cDNAusing a 5′ vector oligo ZC14,063 (SEQ ID NO:8) and 3′ alpha tubulinspecific oligo primer ZC17,574 (SEQ ID NO:9) or 3′ G3PDH specific oligoprimer ZC17,600 (SEQ ID NO:10); and (3) a sample was sent to sequencingto check for possible ribosomal or mitochondrial DNA contamination. Thepanel was set up in a 96-well format that included a human genomic DNA(Clontech, Palo Alto, Calif.) positive control sample. Each wellcontained approximately 0.2-100 pg/μl of cDNA. The PCR was set up usingoligos ZC37685 (SEQ ID NO:26) and ZC37681 (SEQ ID NO:27), TaKaRa Ex Taq™(TAKARA Shuzo Co LTD, Biomedicals Group, Japan), and Rediload dye(Research Genetics, Inc., Huntsville, Ala.). The amplification wascarried out as follows: 1 cycle at 94° C. for 2 minutes, 5 cycles of 94°C. for 30 seconds, 70° C. for 30 seconds, 35 cycles of 94° C. for 30seconds, 64° C. for 30 seconds and 72° C. for 30 seconds, followed by 1cycle at 72° C. for 5 minutes. About 10 μl of the PCR reaction productwas subjected to standard Agarose gel electrophoresis using a 4% agarosegel. The correct predicted DNA fragment size was observed in adrenalgland, bladder, cervix, colon, fetal heart, fetal skin, liver, lung,melanoma, ovary, salivary gland, small intestine, stomach, brain, fetalliver, kidney, prostate, spinal cord, thyroid, placenta, testis, tumoresophagus, tumor liver, tumor ovary, tumor rectum, tumor stomach, tumoruterus, bone marrow, CD3+ library, HaCAT library, HPV library and HPVSlibrary. As this primer pair does not span an intron, there may be riskthat some tissues that are contaminated with genomic DNA or unprocessedmRNA messages would create a false positive in this assay.

Therefore, a different primer pair ZC38481 (SEQ ID NO:47) and ZC38626(SEQ ID NO:48) that span introns were used using the methods describedabove, to re-evaluate the tissue distribution. The correct predicted DNAfragment size (256 bp) was observed in colon, fetal heart, fetal liver,kidney, liver, lung, mammary gland, prostate, salivary gland, smallintestine, adipocyte library, brain library, islet library, and prostatelibrary, RPMI 1788 (B-cell line), spinal cord, placenta library, testis,tumor esophagus, tumor ovary, tumor rectum, tumor stomach, HaCATlibrary, HPV library and HPVS library.

Mouse tissue panels were also examined using another set of primerpairs: (1) ZC38706 (SEQ ID NO:49) and ZC38711 (SEQ ID NO:50) (800 bpproduct) using the methods described above. This panel showed a limitedtissue distribution for mouse zcytor19: mouse prostate cell lines,salivary gland library, and skin.

TABLE 7 Tissue/Cell line #samples Tissue/Cell line #samples Adrenalgland 1 Bone marrow 3 Bladder 1 Fetal brain 3 Bone Marrow 1 Islet 2Brain 1 Prostate 3 Cervix 1 RPMI #1788 2 (ATCC # CCL-156) Colon 1 Testis4 Fetal brain 1 Thyroid 2 Fetal heart 1 WI38 (ATCC # 2 CCL-75 Fetalkidney 1 ARIP (ATCC # 1 CRL-1674 - rat) Fetal liver 1 HaCat - human 1keratinocytes Fetal lung 1 HPV (ATCC # CRL-2221) 1 Fetal muscle 1Adrenal gland 1 Fetal skin 1 Prostate SM 2 Heart 2 CD3+ selected PBMC's1 Ionomycin + PMA stimulated K562 (ATCC 1 HPVS (ATCC # CRL 1 # CCL-243)-2221) - selected Kidney 1 Heart 1 Liver 1 Pituitary 1 Lung 1 Placenta 2Lymph node 1 Salivary gland 1 Melanoma 1 HL60 (ATCC # CCL-240) 3Pancreas 1 Platelet 1 Pituitary 1 HBL-100 1 Placenta 1 Renal mesangial 1Prostate 1 T-cell 1 Rectum 1 Neutrophil 1 Salivary Gland 1 MPC 1Skeletal muscle 1 Hut-102 (ATCC # 1 TIB-162) Small intestine 1Endothelial 1 Spinal cord 1 HepG2 (ATCC # 1 HB-8065) Spleen 1 Fibroblast1 Stomach 1 E. Histo 1 Testis 2 Thymus 1 Thyroid 1 Trachea 1 Uterus 1Esophagus tumor 1 Gastric tumor 1 Kidney tumor 1 Liver tumor 1 Lungtumor 1 Ovarian tumor 1 Rectal tumor 1 Uterus tumor 1

Example 3 PCR-Based Chromosomal Mapping of the Zcytor19 Gene

Zcytor19 is mapped to chromosome 1 using the commercially available“GeneBridge 4 Radiation Hybrid (RH) Mapping Panel” (Research Genetics,Inc., Huntsville, Ala.). The GeneBridge 4 RH panel contains DNA fromeach of 93 radiation hybrid clones, plus two control DNAs (the HFL donorand the A23 recipient). A publicly available WWW server(http://www-genome.wi.mitedu/cgi-bin/contig/rhmapper.pl) allows mappingrelative to the Whitehead Institute/MIT Center for Genome Research'sradiation hybrid map of the human genome (the “WICGR” radiation hybridmap) which is constructed with the GeneBridge 4 RH panel.

For the mapping of Zcytor19 with the GeneBridge 4 RH panel, 20 μlreactions are set up in a 96-well microtiter plate compatible for PCR(Stratagene, La Jolla, Calif.) and used in a “RoboCycler Gradient 96”thermal cycler (Stratagene). Each of the 95 PCR reactions consisted of 2μl 10× KlenTaq PCR reaction buffer (CLONTECH Laboratories, Inc., PaloAlto, Calif.), 1.6 μl dNTPs mix (2.5 mM each, PERKIN-ELMER, Foster City,Calif.), 1 μl sense primer, ZC27,895 (SEQ ID NO:14), 1 μl antisenseprimer, ZC27,899 (SEQ ID NO:24), 2 μl “RediLoad” (Research Genetics,Inc., Huntsville, Ala.), 0.4 μl 50× Advantage KlenTaq Polymerase Mix(Clontech Laboratories, Inc.), 25 ng of DNA from an individual hybridclone or control and distilled water for a total volume of 20 μl. Thereactions are overlaid with an equal amount of mineral oil and sealed.The PCR cycler conditions are as follows: an initial 1 cycle 5 minutedenaturation at 94° C., 35 cycles of a 45 seconds denaturation at 94°C., 45 seconds annealing at 54° C. and 1 minute AND 15 seconds extensionat 72° C., followed by a final 1 cycle extension of 7 minutes at 72° C.The reactions are separated by electrophoresis on a 2% agarose gel (EMScience, Gibbstown, N.J.) and visualized by staining with ethidiumbromide. The results show that Zcytor19 maps on the chromosome 1 WICGRradiation hybrid map in the 1p36.11 chromosomal region.

Example 4 Construction of Mammalian Expression Vectors that ExpressZcytor19 Soluble Receptors Zcytor19CEE, Zcytor19CFLG, Zcytor19CHIS andZcytor19-Fc4 A. Construction of Zcytor19 Mammalian Expression VectorContaining Zcytor19CEE, Zcytor19CFLG and Zcytor19CHIS

An expression vector is prepared for the expression of the soluble,extracellular domain of the zcytor19 polypeptide, pC4zcytor19CEE,wherein the construct is designed to express a zcytor19 polypeptidecomprised of the predicted initiating methionine and truncated adjacentto the predicted transmembrane domain, and with a C-terminal Glu-Glu tag(SEQ ID NO:11).

A zcytor19 DNA fragment comprising a zcytor19 extracellular or cytokinebinding domain of zcytor19 described herein, is created using PCR, andpurified using standard methods. The excised DNA is subcloned into aplasmid expression vector that has a signal peptide, e.g., the nativezcytor19 signal peptide, and attaches a Glu-Glu tag (SEQ ID NO:11) tothe C-terminus of the zcytor19 polypeptide-encoding polynucleotidesequence. Such a mammalian expression vector contains an expressioncassette having a mammalian promoter, multiple restriction sites forinsertion of coding sequences, a stop codon and a mammalian terminator.The plasmid can also have an E. coli origin of replication, a mammalianselectable marker expression unit having an SV40 promoter, enhancer andorigin of replication, a DHFR gene and the SV40 terminator.

Restriction digested zcytor19 insert and previously digested vector areligated using standard molecular biological techniques, andelectroporated into competent cells such as DH10B competent cells (GIBCOBRL, Gaithersburg, Md.) according to manufacturer's direction and platedonto LB plates containing 50 mg/ml ampicillin, and incubated overnight.Colonies are screened by restriction analysis of DNA prepared fromindividual colonies. The insert sequence of positive clones is verifiedby sequence analysis. A large scale plasmid preparation is done using aQIAGEN® Maxi prep kit (Qiagen) according to manufacturer's instructions.

The same process is used to prepare the zcytor19 soluble receptors witha C-terminal his tag, composed of 6 His residues in a row; and aC-terminal FLAG® tag (SEQ ID NO:12), zcytor19CFLAG. To construct theseconstructs, the aforementioned vector has either the CHIS or the FLAG®tag in place of the glu-glu tag (SEQ ID NO:11).

B. Mammalian Expression Construction of Soluble Human Zcytor19 Receptor:Zcytor19-Fc4

An expression vector, zcytor19/Fc4/pzmp20, was prepared to express aC-terminally Fc4 tagged soluble version of zcytor19 (human zcytor19-Fc4)in BHK cells. A fragment of zcytor19 cDNA that includes thepolynucleotide sequence from extracellular domain of the zcytor19receptor was fused in frame to the Fc4 polynucleotide sequence (SEQ IDNO:13) to generate a zcytor19-Fc4 fusion (SEQ ID NO:22 and SEQ IDNO:23). The pzmp20 vector is a mammalian expression vector that containsthe Fc4 polynucleotide sequence and a cloning site that allows rapidconstruction of C-terminal Fc4 fusions using standard molecular biologytechniques.

A 630 base pair fragment was generated by PCR, containing theextracellular domain of human zcytor19 with BamHI and Bgl2 sites codedon the 5′ and 3′ ends, respectively. This PCR fragment was generatedusing primers ZC37967 (SEQ ID NO:24) and ZC37972 (SEQ ID NO:25) byamplification from human brain cDNA library. The PCR reaction conditionswere as follows: 30 cycles of 94° C. for 20 seconds, and 68° C. for 2minutes; 1 cycle at 68° C. for 4 minutes; followed by a 10° C. soak. Thefragment was digested with BamHI and Bgl2 restriction endonucleases andsubsequently purified by 1% gel electrophoresis and band purificationusing QiaQuick gel extraction kit (Qiagen). The resulting purified DNAwas ligated for 5 hours at room temperature into a pzmp20 vectorpreviously digested with Bgl2 containing Fc4 3′ of the Bgl2 sites.

One μl of the ligation mix was electroporated in 37 μl DH10Belectrocompetent E. coli (Gibco) according to the manufacturer'sdirections. The transformed cells were diluted in 400 μl of LB media andplated onto LB plates containing 100 μg/ml ampicillin. Clones wereanalyzed by restriction digests and positive clones were sent for DNAsequencing to confirm the sequence of the fusion construct.

Example 5 Transfection and Expression of Zcytor19 Soluble ReceptorPolypeptides A. Mammalian Expression Human Zcytor19 Soluble Receptor:Zcytor19/Fc4

BHK 570 cells (ATCC NO: CRL-10314) were plated in T-75 tissue cultureflasks and allowed to grow to approximately 50 to 70% confluence at 37°C., 5% CO₂, in DMEM/FBS media (DMEM, Gibco/BRL High Glucose, (Gibco BRL,Gaithersburg, Md.), 5% fetal bovine serum, 1 mM L-glutamine (JRHBiosciences, Lenea, Kans.), 1 mM sodium pyruvate (Gibco BRL)). The cellswere then transfected with the plasmid zcytor19/Fc4/pzmp20 (Example 4B)using Lipofectamine™ (Gibco BRL), in serum free (SF) media formulation(DMEM, 10 mg/ml transferrin, 5 mg/ml insulin, 2 mg/ml fetuin, 1%L-glutamine and 1% sodium pyruvate). Ten μg of the plasmid DNAzcytor19/Fc4/pzmp20 (Example 4B) was diluted into a 15 ml tube to atotal final volume of 500 μl with SF media. 50 μl of Lipofectamine wasmixed with 450 μl of SF medium. The Lipofectamine mix was added to theDNA mix and allowed to incubate approximately 30 minutes at roomtemperature. Four ml of SF media was added to the DNA:Lipofectaminemixture. The cells were rinsed once with 5 ml of SF media, aspirated,and the DNA:Lipofectamine mixture was added. The cells were incubated at37° C. for five hours, and then 5 ml of DMEM/10% FBS media was added.The flask was incubated at 37° C. overnight after which time the cellswere split into the selection media (DMEM/FBS media from above with theaddition of 1 μM methotrexate (Sigma Chemical Co., St. Louis, Mo.) in150 mm plates at 1:2, 1:10, and 1:50. Approximately 10 dayspost-transfection, one 150 mm plate of 1 μM methotrexate resistantcolonies was trypsinized, the cells were pooled, and one-half of thecells were replated in 10 μM methotrexate; to further amplify expressionof the zcytor19/Fc4 protein. A conditioned-media sample from this poolof amplified cells was tested for expression levels using SDS-PAGE andWestern analysis.

Single clones expressing the soluble receptors can also isolated,screened and grown up in cell culture media, and purified using standardtechniques. Moreover, CHO cells are also suitable cells for suchpurposes.

Example 6 Assessing Zcytor19 Receptor Heterodimerization Using ORIGENAssay

Soluble zcytor19 receptor zcytor19CFLAG (Example 4 and Example 5), orgp130 (Hibi, M. et al., Cell 63:1149-1157, 1990) are biotinylated byreaction with a five-fold molar excess of sulfo-NHS-LC-Biotin (Pierce,Inc., Rockford, Ill.) according to the manufacturer's protocol. Solublezcytor19 receptor and another soluble receptor subunit, for example,soluble class II cytokine receptors, for example, interferon-gamma,alpha and beta chains and the interferon-alpha/beta receptor alpha andbeta chains, zcytor11 (commonly owned U.S. Pat. No. 5,965,704), CRF2-4(SEQ ID NO:64), DIRS1, zcytor7 (commonly owned U.S. Pat. No. 5,945,511)soluble receptors. Receptors in this subfamily may associate to formheterodimers that transduce a signal. These soluble receptors arelabeled with a five fold molar excess of Ru-BPY-NHS (Igen, Inc.,Gaithersburg, Md.) according to manufacturer's protocol. Thebiotinylated and Ru-BPY-NHS-labeled forms of the soluble zcytor19receptor can be respectively designated Bio-zcytor19 receptor andRu-zcytor19; the biotinylated and Ru-BPY-NHS-labeled forms of the othersoluble receptor subunit can be similarly designated. Assays can becarried out using conditioned media from cells expressing a ligand thatbinds zcytor19 heterodimeric receptors, or using purified ligands.Preferred ligands are zcyto20 (SEQ ID NO:52), zcyto21 (SEQ ID NO:55),zcyto22 (SEQ ID NO:57), zcyto24 (SEQ ID NO:60), zcyto25 (SEQ ID NO:62),and ligands that can bind class II heterodimeric cytokine receptors suchas, IL-10, IL-9, IL-TIF, interferons, TSLP (Levine, S D et al., ibid.;Isaksen, D E et al., ibid.; Ray, R J et al., ibid.; Friend, S L et al.,ibid.), and the like.

For initial soluble receptor binding characterization, the cytokinesmentioned above, or conditioned medium, are tested to determine whetherthey can mediate homodimerization of zcytor19 receptor and if they canmediate the heterodimerization of zcytor19 receptor with the solublereceptor subunits described above. To do this, 50 μl of conditionedmedia or TBS-B containing purified cytokine, is combined with 50 μl ofTBS-B (20 mM Tris, 150 mM NaCl, 1 mg/ml BSA, pH 7.2) containing e.g.,400 ng/ml of Ru-zcytor19 receptor and Bio-zcytor19, or 400 ng/ml ofRu-zcytor19 receptor and e.g., Bio-CRF2-4, or 400 ng/ml of e.g.,Ru-CRF2-4 and Bio-zcytor19. Following incubation for one hour at roomtemperature, 30 μg of streptavidin coated, 2.8 mm magnetic beads (Dynal,Inc., Oslo, Norway) are added and the reaction incubated an additionalhour at room temperature. 200 μl ORIGEN assay buffer (Igen, Inc.,Gaithersburg, Md.) is then added and the extent of receptor associationmeasured using an M8 ORIGEN analyzer (Igen, Inc.).

Example 7 Construct for Generating a Zcytor19 Receptor Heterodimer

A vector expressing a secreted human zcytor19 heterodimer isconstructed. In this construct, the extracellular cytokine-bindingdomain of zcytor19 is fused to the heavy chain of IgG gamma 1 (IgGγ1)(SEQ ID NO:14 and SEQ ID NO:15), while the extracellular portion of theheteromeric cytokine receptor subunit (E.g., class II cytokinereceptors, for example, CRF2-4) is fused to a human kappa light chain(human κ light chain) (SEQ ID NO:16 and SEQ ID NO:17).

A. Construction of IgG Gamma 1 and Human κ Light Chain Fusion Vectors

The heavy chain of IgGγ1 (SEQ ID NO:14) is cloned into the Zem229Rmammalian expression vector (ATCC deposit No. 69447) such that anydesired cytokine receptor extracellular domain having a 5′ EcoRI and 3′NheI site can be cloned in resulting in an N-terminal extracellulardomain-C-terminal IgGγ1 fusion. The IgGγ1 fragment used in thisconstruct is made by using PCR to isolate the IgGγ1 sequence from aClontech hFetal Liver cDNA library as a template. PCR products arepurified using methods described herein and digested with MluI and EcoRI(Boerhinger-Mannheim), ethanol precipitated and ligated with oligos thatcomprise an MluI/EcoRI linker, into Zem229R previously digested with andEcoRI using standard molecular biology techniques disclosed herein.

The human κ light chain (SEQ ID NO:16) is cloned in the Zem228Rmammalian expression vector (ATCC deposit No. 69446) such that anydesired cytokine receptor extracellular domain having a 5′ EcoRI siteand a 3′ KpnI site can be cloned in resulting in a N-terminal cytokineextracellular domain-C-terminal human κ light chain fusion. As a KpnIsite is located within the human κ light chain sequence (cleaved by theKpnI enzyme after nucleotide 62 in SEQ ID NO:16), a special primer isdesigned to clone the 3′ end of the desired extracellular domain of acytokine receptor into this KpnI site: The primer is designed so thatthe resulting PCR product contains the desired cytokine receptorextracellular domain with a segment of the human κ light chain up to theKpnI site (SEQ ID NO:16). This primer preferably comprises a portion ofat least 10 nucleotides of the 3′ end of the desired cytokine receptorextracellular domain fused in frame 5′ to SEQ ID NO:16. The human κlight chain fragment used in this construct is made by using PCR toisolate the human κ light chain sequence from the same Clontech humanFetal Liver cDNA library used above. PCR products are purified usingmethods described herein and digested with MluI and EcoRI(Boerhinger-Mannheim), ethanol precipitated and ligated with theMluI/EcoRI linker described above, into Zem228R previously digested withand EcoRI using standard molecular biology techniques disclosed herein.

B. Insertion of Zcytor19 Receptor or Heterodimeric Subunit ExtracellularDomains into Fusion Vector Constructs

Using the construction vectors above, a construct having zcytor19 fusedto IgGγ1 is made. This construction is done by PCRing the extracellulardomain or cytokine-binding domain of zcytor19 receptor described hereinfrom a prostate cDNA library (Clontech) or activated lymphocyte cDNAlibrary using standard methods, and oligos that provide EcoRI and NheIrestriction sites. The resulting PCR product is digested with EcoRI andNheI, gel purified, as described herein, and ligated into a previouslyEcoRI and NheI digested and band-purified Zem229R/IgGγ1 described above.The resulting vector is sequenced to confirm that the zcytor19/IgG gamma1 fusion (zcytor19/Ch1 IgG) is correct.

A separate construct having a heterodimeric cytokine receptor subunitextracellular domain, i.e., CRF2-4 (SEQ ID NO: 64) fused to κ light isalso constructed as above. The cytokine receptor/human κ light chainconstruction is performed as above by PCRing from, e.g., a lymphocytecDNA library (Clontech) using standard methods, and oligos that provideEcoRI and KpnI restriction sites. The resulting PCR product is digestedwith EcoRI and KpnI and then ligating this product into a previouslyEcoRI and KpnI digested and band-purified Zem228R/human κ light chainvector described above. The resulting vector is sequenced to confirmthat the cytokine receptor subunit/human κ light chain fusion iscorrect.

D. Co-Expression of the Zcytor19 and Heterodimeric Cytokine ReceptorSubunit Extracellular Domain

Approximately 15 μg of each of vectors above, are co-transfected intomammalian cells, e.g., BHK-570 cells (ATCC No. CRL-10314) usingLipofectaminePlus™ reagent (Gibco/BRL), as per manufacturer'sinstructions. The transfected cells are selected for 10 days in DMEM+5%FBS (Gibco/BRL) containing 1 μM of methotrexate (MTX) (Sigma, St. Louis,Mo.) and 0.5 mg/ml. G418 (Gibco/BRL) for 10 days. The resulting pool oftransfectants is selected again in 10 μm of MTX and 0.5 mg/ml G418 for10 days.

The resulting pool of doubly selected cells is used to generate protein.Three Factories (Nunc, Denmark) of this pool are used to generate 10 Lof serum free conditioned medium. This conditioned media is passed overa 1 ml protein-A column and eluted in about 10, 750 microliterfractions. The fractions having the highest protein concentration arepooled and dialyzed (10 kD MW cutoff) against PBS. Finally the dialyzedmaterial is submitted for amino acid analysis (AAA) using routinemethods.

Example 8 Reconstitution of Zcytor19 Receptor In Vitro

To identify components involved in the zcytor19-signaling complex,receptor reconstitution studies are performed as follows. For example,BHK 570 cells (ATCC No. CRL-10314) transfected, using standard methodsdescribed herein, with a luciferase reporter mammalian expression vectorplasmid serve as a bioassay cell line to measure signal transductionresponse from a transfected zcytor19 receptor complex to the luciferasereporter in the presence of zcytor19 Ligand. MK cells would be used inthe event that BHK cells do not endogenously express the zcytor19receptor. Other cell lines can be used. An exemplary luciferase reportermammalian expression vector is the KZ134 plasmid which is constructedwith complementary oligonucleotides that contain STAT transcriptionfactor binding elements from 4 genes. A modified c-fos Sis inducibleelement (m67SIE, or hSIE) (Sadowski, H. et al., Science 261:1739-1744,1993), the p21 SIE1 from the p21 WAF1 gene (Chin, Y. et al., Science272:719-722, 1996), the mammary gland response element of the β-caseingene (Schmitt-Ney, M. et al., Mol. Cell. Biol. 11:3745-3755, 1991), anda STAT inducible element of the Fcg RI gene, (Seidel, H. et al., Proc.Natl. Acad. Sci. 92:3041-3045, 1995). These oligonucleotides containAsp718-XhoI compatible ends and are ligated, using standard methods,into a recipient firefly luciferase reporter vector with a c-Fospromoter (Poulsen, L. K. et al., J. Biol. Chem. 273:6229-6232, 1998)digested with the same enzymes and containing a neomycin selectablemarker. The KZ134 plasmid is used to stably transfect BHK, or BaF3cells, using standard transfection and selection methods, to make aBHK/KZ134 or BaF3/KZ134 cell line respectively.

The bioassay cell line is transfected with zcytor19 receptor alone, orco-transfected with zcytor19 receptor along with one of a variety ofother known receptor subunits. Receptor complexes include but are notlimited to zcytor19 receptor only, various combinations of zcytor19receptor with class II cytokine receptors, for example,interferon-gamma, alpha and beta chains and the interferon-alpha/betareceptor alpha and beta chains, zcytor11 (commonly owned U.S. Pat. No.5,965,704), CRF2-4, DIRS1, zcytor7 (commonly owned U.S. Pat. No.5,945,511) receptors. Each independent receptor complex cell line isthen assayed in the presence of cytokine-conditioned media or purifiedcytokines and luciferase activity measured using routine methods. Theuntransfected bioassay cell line serves as a control for the backgroundluciferase activity, and is thus used as a baseline to compare signalingby the various receptor complex combinations. The conditioned medium orcytokine that binds the zcytor19 receptor in the presence of the correctreceptor complex, is expected to give a luciferase readout ofapproximately 5 fold over background or greater.

As an alternative, a similar assay can be performed wherein the aBaf3/zcytor19 cell line is co-transfected as described herein andproliferation is measured, using a known assay such as a standard AlamarBlue proliferation assay.

Example 9

A: COS Cell Transfection and Secretion Trap

Biotinylated zcyto21 (SEQ ID NO:55) was tested for binding to known ororphan cytokine receptors. The pZP7 expression vectors containing cDNAsof cytokine receptors (including human IFNαR1, IFNβR1, IFNαR2, IFNβR2,IL-10R, CRF2-4, ZcytoR7, DIRS1, Zcytor19, and Tissue Factor) weretransfected into COS cells, and the binding of biotinylated zcyto20 totransfected COS cells was carried out using the secretion trap assaydescribed below. Positive binding in this assay showed receptor-ligandpairs.

COS Cell Transfections

The COS cell transfections were performed as follows: COS cells wereplated (1×10⁵ cells/well) on fibronectin coated, 12-well, tissue cultureplates (Becton Dickinson, Bedford, Mass.) and incubated at 37° C.overnight. Cytokine receptor DNA (0.75 μg) was mixed with 50 μl serumfree DMEM media (55 mg sodium pyruvate, 146 mg L-glutamine, 5 mgtransferrin, 2.5 mg insulin, 1 μg selenium and 5 mg fetuin in 500 mlDMEM), then mixed with 5 μl Lipofectamine™ (Invitrogen, Carlsbad,Calif.) in 45 μl serum free DMEM media, and incubated at roomtemperature for 30 minutes. An additional 400 μl serum free DMEM mediawas added. The cells were rinsed with serum free DMEM, and 5000 of theDNA mixture was added. The cells were incubated for 5 hours at 37° C.,at which time an additional 500 μl 20% FBS DMEM media (100 ml FBS, 55 mgsodium pyruvate and 146 mg L-glutamine in 500 ml DMEM) was added and thecells were incubated overnight.

Secretion Trap Assay

The secretion trap was performed as follows: Media was aspirated andcells were rinsed twice with 1% BSA in PBS. Cells were blocked for 1hour with TNB (0.1M Tris-HCL, 0.15M NaCl and 0.5% Blocking Reagent (NENRenaissance TSA-Direct Kit, NEN Life Science Products, Boston, Mass.) inH₂O. The cells were incubated for 1 hour with 3 μg/ml biotinylatedzcyto21 protein (Example 27) in TNB. Cells were then washed 3 times with1% BSA in PBS and were incubated for another hour with 1:300 dilutedStreptavidin-HRP (NEN kit) in TNB. Again cells were washed 3 times with1% BSA in PBS, and then fixed for 15 minutes with 1.8% Formaldehyde inPBS. Cells were then washed 3 times with TNT (0.1M Tris-HCL, 0.15M NaCl,and 0.05% Tween-20 in H₂O).

Positive binding was detected with fluorescein tyramide reagent diluted1:50 in dilution buffer (NEN kit), incubated for 4.5 minutes, and washedwith TNT. Cells were preserved with Vectashield Mounting Media (VectorLabs Burlingame, Calif.) diluted 1:5 in TNT. Cells were visualized usinga FITC filter on fluorescent microscope.

Positive binding was detected on cells transfected with human zcytor19cDNA and incubated with biotinylated zcyto21. None of the othertransfected receptors bound zcyto21, and zcytor19 did not bind a controlbiotinylated protein. These data indicate that zcytor19 is a receptorfor zcyto21.

Further experiments have shown positive binding between both human andmouse Zcytor19 with biotinylated zcyto21. Positive binding was alsodetected on cells transfected with human zcytor19 cDNA and incubatedwith biotinylated zcyto20, and zcyto24.

Example 10 Expression of Human Zcytor19 in E. coli

A. Construction of Zcytor19-MBP Fusion Expression VectorpTAP170/Zcytor19

An expression plasmid containing a polynucleotide encoding part of thehuman zcytor19 fused N-terminally to maltose binding protein (MBP) wasconstructed via homologous recombination. A fragment of human zcytor19cDNA (SEQ ID NO:1) was isolated using PCR. Two primers were used in theproduction of the human zcytor19 fragment in a PCR reaction: (1) PrimerZC39204 (SEQ ID NO:30), containing 40 bp of the vector flanking sequenceand 24 bp corresponding to the amino terminus of the human zcytor19, and(2) primer ZC39205 (SEQ ID NO:31), containing 40 bp of the 3′ endcorresponding to the flanking vector sequence and 24 bp corresponding tothe carboxyl terminus of the human zcytor19. The PCR reaction conditionswere as follows: 1 cycle of 94 C for 1 minute. Then 20 cycles of 94° C.for 30 seconds, 60° C. for 30 seconds, and 68° C. for 1.5 minutes;followed by 4° C. soak, run in duplicate. Five μl of each 100 μl PCRreaction were run on a 1.0% agarose gel with 1×TBE buffer for analysis,and the expected band of approximately 700 bp fragment was seen. Theremaining 95 μl of PCR reaction was combined with the second PCR tubeprecipitated with 400 μl of absolute ethanol and resuspended in 141 ofwater to be used for recombining into the Sma1 cut recipient vectorpTAP170 to produce the construct encoding the MBP-human zcytor19 fusion,as described below.

Plasmid pTAP170 was derived from the plasmids pRS316 and pMAL-c2. Theplasmid pRS316 is a Saccharomyces cerevisiae shuttle vector (Hieter P.and Sikorski, R., Genetics 122:19-27, 1989). pMAL-C2 (NEB) is an E. coliexpression plasmid. It carries the tac promoter driving MalE (geneencoding MBP) followed by a His tag, a thrombin cleavage site, a cloningsite, and the rrnB terminator. The vector pTAP170 was constructed usingyeast homologous recombination. 100 ng of EcoR1 cut pMAL-c2 wasrecombined with 1 μg Pvu1 cut pRS316, 1 μg linker, and 1 μg Sca1/EcoR1cut pRS316. The linker consisted of oligos zc19,372 (100 pmole):zc19,351 (1 pmole): zc19,352 (1 pmole), and zc19,371 (100 pmole)combined in a PCR reaction. Conditions were as follows: 10 cycles of 94°C. for 30 seconds, 50° C. for 30 seconds, and 72° C. for 30 seconds;followed by 4° C. soak. PCR products were concentrated via 100% ethanolprecipitation.

One hundred microliters of competent yeast cells (S. cerevisiae) werecombined with 10 μl of a mixture containing approximately 1 μg of thehuman zcytor19 insert, and 100 ng of SmaI digested pTAP170 vector, andtransferred to a 0.2 cm electroporation cuvette. The yeast/DNA mixturewas electropulsed at 0.75 kV (5 kV/cm), infinite ohms, 25 μF. To eachcuvette was added 600 μl of 1.2 M sorbitol. The yeast was then plated intwo 300 μl aliquots onto two -URA D plates and incubated at 30° C.

After about 48 hours, the Ura+ yeast transformants from a single platewere resuspended in 1 ml H₂O and spun briefly to pellet the yeast cells.The cell pellet was resuspended in 1 ml of lysis buffer (2% TritonX-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). Five hundredmicroliters of the lysis mixture was added to an Eppendorf tubecontaining 300 μl acid washed glass beads and 500 μl phenol-chloroform,vortexed for 1 minute intervals two or three times, followed by a 5minute spin in a Eppendorf centrifuge at maximum speed. Three hundredmicroliters of the aqueous phase was transferred to a fresh tube, andthe DNA precipitated with 600 μl ethanol (EtOH), followed bycentrifugation for 10 minutes at 4° C. The DNA pellet was resuspended in100 μl H₂O.

Transformation of electrocompetent E. coli cells (MC1061, Casadaban et.al. J. Mol. Biol. 138, 179-207) was done with 1 μl yeast DNA prep and 40μl of MC1061 cells. The cells were electropulsed at 2.0 kV, 25 μF and400 ohms. Following electroporation, 0.6 ml SOC (2% Bactol Tryptone(Difco, Detroit, Mich.), 0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mMKCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM glucose) was added to the cells.After incubation for one hour at 37° C., the cells were plated in onealiquot on LB Kan plates (LB broth (Lennox), 1.8% Bacto™ Agar (Difco),30 mg/L kanamycin).

Individual clones harboring the correct expression construct for humanzcytor19 were identified by expression. Cells were grown in SuperbrothII (Becton Dickinson) with 30 μg/ml of kanamycin overnight. 50 μl of theovernight culture was used to inoculate 2 ml of fresh Superbroth II+30μg/ml kanamycin. Cultures were grown at 37° C., shaking for 2 hours. 1ml of the culture was induced with 1 mM IPTG. 2-4 hours later the 250 μlof each culture was mixed 250 μl Thorner buffer with 5% βME and dye (8Murea, 100 mM Tris pH7.0, 10% glycerol, 2 mM EDTA, 5% SDS). Samples wereboiled for 5-10 minutes. 20 μl were loaded per lane on a 4%-12% PAGE gel(NOVEX). Gels were run in 1×MES buffer. The positive clones weredesignated pTAP317 and subjected to sequence analysis. Thepolynucleotide sequence of MBP-zcytor19 fusion within pTAP317 is shownin SEQ ID NO:32, and the corresponding polypeptide sequence of theMBP-zcytor19 fusion is shown in SEQ ID NO:33.

B. Bacterial Expression of Human Zcytor19.

Ten microliters of sequencing DNA was digested with NotI (NEB) in thefollowing reaction to remove the CEN-ARS: 10 μl DNA, 3 μl buffer3 (NEB),15 μl water, and 2 μl NotI (10 U/μl NEB) at 37° C. for one hour. Then 7μl of the digest was mixed with 2 μl of 5× buffer and T4DNA ligase(1u/μl BRL). Reaction was incubated at room temperature for one hour.One microliter of the reaction was transformed into the E. coli strainW3110 (ATCC). The cells were electropulsed at 2.0 kV, 25 μF and 400ohms. Following electroporation, 0.6 ml SOC (2% Bacto™ Tryptone (Difco,Detroit, Mich.), 0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl, 10mM MgCl2, 10 mM MgSO4, 20 mM glucose) was added to the cells. After aone hour incubation at 37° C., the cells were plated in one aliquot onLB Kan plates (LB broth (Lennox), 1.8% Bacto™ Agar (Difco), 30 mg/LKanamycin). Individual clones were analyzed by diagnostic digests forthe absence of yeast marker and replication sequence.

A positive clone was used to inoculate an overnight starter culture ofSuperbroth II (Becton Dickinson) with 30 μg/ml of kanamycin. The starterculture was used to inoculate 4 2 L-baffled flasks each filled with 500ml of Superbroth II+Kan. Cultures shook at 37° C. at 250 rpm until theOD₆₀₀ reached 4.1. At this point, the cultures were induced with 1mMIPTG. Cultures grew for two more hours at 37° C., 250 rpm at whichpoint 2 ml was saved for analysis and the rest was harvested viacentrifugation. Pellet was saved at −80° C. until transferred to proteinpurification.

Example 11 Purification Scheme for Zcytor19-FC4 Fusion

All procedures performed at 4 C, unless otherwise noted. The conditionedmedia was concentrated first 20 times by using an Amicon/MilliporeSpiral cartridge, 10 kD MWCO. (at ambient temperature) The concentratedmedia was then applied to an appropriately sized POROS 50 A (coupledprotein A) column at an optimal capture flow rate. The column was washedwith 10 column volumes (CV) of equilibration buffer, then rapidly elutedwith 3 CV of 0.1 M Glycine pH 3. The collected fractions had apredetermined volume of 2M TRIS pH 8.0 added prior to the elution toneutralize the pH to about 7.2.

Brilliant Blue (Sigma) stained NuPAGE gels were ran to analyze theelution. Fractions of interested were pooled and concentrated using a 30kD MWCO centrifugal concentrator to a nominal volume. The concentratedProtein A pool was injected onto an appropriately sized PhamiciaSephacryl 200 column to remove aggregates and to buffer exchange theprotein into PBS pH 7.3. Brilliant Blue (Sigma) stained NuPAGE gels wereagain used to analyze the elution. Fractions were pooled. Western andBrilliant Blue (Sigma) stained NuPAGE gels were ran to confirm purityand content. For further analysis, the protein was submitted for AAA,and N-terminal sequencing. AAA analysis and N-terminal sequencingverified the zcytor19-Fc polyepptide; the N-terminal amino acid sequencewas as expected SRPRL APPQX VTLLS QNFSV (SEQ ID NO:34).

Example 12 Human or 19 Expression Based on RT-PCR Analysis of MultipleTissue and Blood Fraction First-Strand cDNA Panels

Gene expression of zcytor19 was examined using commercially availablenormalized multiple tissue first-strand cDNA panels (OriGeneTechnologies, Inc. Rockville, Md.; BD Biosciences Clontech, Palo Alto,Calif.). These included OriGene's Human Tissue Rapid-Scan™ Panel(containing 24 different tissues) and the following BD BiosciencesClontech Multiple Tissue cDNA (MTC™) Panels: Human MTC Panel I(containing 8 different adult tissues), Human MTC Panel II (containing 8different adult tissues), Human Fetal MTC Panel (containing 8 differentfetal tissues), Human Tumor MTC Panel (containing carcinomas from 7different organs), Human Blood Fractions MTC Panel (containing 9different blood fractions), and Human Immune System MTC Panel(containing 6 different organs and peripheral blood leukocyte).

PCR reactions were set up using zcytor19 specific oligo primers ZC40285(SEQ ID NO:35) and ZC40286 (SEQ ID NO:36) which yield a 426 bp product,Qiagen HotStarTaq DNA Polymerase (Qiagen, Inc., Valencia, Calif.) andRediLoad™ dye (Research Genetics, Inc., Huntville, Ala.). The PCR cyclerconditions were as follows: an initial 1 cycle 15 minute denaturation at95° C., 35 cycles of a 45 second denaturation at 95° C., 1 minuteannealing at 63° C. and 1 minute and 15 seconds extension at 72° C.,followed by a final 1 cycle extension of 7 minutes at 72° C. Thereactions were separated by electrophoresis on a 2% agarose gel (EMScience, Gibbstown, N.J.) and visualized by staining with ethidiumbromide.

A DNA fragment of the correct size was observed in the following humanadult tissues: adrenal gland, bone marrow, colon, heart, liver, lung,lymph node, muscle, ovary, pancreas, placenta, prostate, salivary gland,small intestine, spleen, stomach, testis, thyroid, and tonsil. A DNAfragment of the correct size was observed in the following human fetaltissues: heart, liver, lung, kidney, skeletal muscle, spleen, andthymus. A DNA fragment of the correct size was observed in the followinghuman blood fractions: peripheral blood leukocyte, mononuclear cells(B-cells, T-cells, and monocytes), resting CD8+ cells(T-suppressor/cytotoxic), resting CD19+ cells (B-cells), activated CD19+cells, activated mononuclear cells, and activated CD4+ cells. A DNAfragment of the correct size was observed in the following tumortissues: breast carcinoma, colon adenocarcinoma, lung carcinoma, ovariancarcinoma, pancreatic adenocarcinoma, and prostatic adenocarcinoma.

Because zcytor19 is expressed in these specific tumor tissues, zcytor19polynucleotides, polypeptides and antibodies can be used as a tumormarker as disclosed herein. Moreover, an antibody to zcytor19 could haveanti-tumor activity, as well as toxin-conjugates, cytokine conjugates orother conjugates of an antibody, or the zcytor19 receptor ligand itself.The antagonist of zcytor19 ligand, such as anti-zcytor19 antibodies orsoluble receptors can also act as anti-tumor reagents.

Example 13 Generation and Analysis of Zcytor19 KO Mice A. Identificationof BAC Clones Positive for Mouse Zcytor19 Gene

One BAC clone positive for mouse zcytor19 gene was identified usingIncyte Genomic's (St. Louis, Mo.) Easy-to-Screen DNA Pools, BAC Mouse ES(Release I) following Manufacturer's instructions. Oligonucleotides weredesigned to generate a PCR fragment containing partial exon 6, completeintron 6 and partial exon 7 sequences.

PCR reactions were carried out in 25 μl using 1.75 units of Advantage 2polymerase (Clontech). Either 2 μl or 10 μl of BAC library DNA was usedas template in buffer containing 67 mM Tris pH 8.8, 16.6 mM (NH₄)₂SO₄,6.7 mM MgCl₂

5 mM 2-Mercaptoethanol, 100 μg/ml gelatin, 10% Dimethyl Sulfoxide, 1 mMdeoxynucleotides, 140 nM forward primer ZC39128 (SEQ ID NO:37) and 140nM reverse primer ZC39129 (SEQ ID NO:38). PCR conditions were as follows95° C. for 1 min; 30 cycles of 95° C. for 15 seconds, 55° C. for 30seconds, and 68° C. for 30 seconds; and 68° C. for 2 minutes; followedby a 4° C. hold. PCR products were analyzed by agarose gelelectrophoresis. Positive PCR products were found to be 1,149 bp.

Four additional BAC clones positive for mouse zcytor19 gene wereidentified using Incyte's BAC Mouse Filter Set (Release II) followingManufacturer's instructions. Oligonucleotides were designed to generatea PCR fragment containing partial exon 6, and partial exon 7 sequencesfrom mouse cDNA template.

PCR reactions were carried out in 25 μl using 1.75 units of Advantage 2polymerase (Clontech). 2 μl of Neonatal Mouse skin cDNA library (JAK062700B) was used as template in buffer containing 67 mM Tris pH 8.8,16.6 mM (NH₄)₂SO₄, 6.7 mM MgCl₂

5 mM 2-Mercaptoethanol, 100 μg/ml gelatin, 10% Dimethyl Sulfoxide, 1 mMdeoxynucleotides, 140 nM forward primer ZC39128 (SEQ ID NO:37) and 140nM reverse primer ZC39129 (SEQ ID NO:38). PCR conditions were asdescribed above. PCR products were separated by agarose gelelectrophoresis and purified using Qiaquick (Qiagen) gel extraction kit.The isolated, approximately 400 bp, DNA fragment was labeled usingPrime-It II (Stratagene) Random Primer labeling kit and purified usingMicroSpin S-200HR columns (AmershamPharmacia).

The labeled probe was used to screen Incyte's 7 filter BAC library set.Hybridizations were carried out at 55° C. overnight using ExpressHyb(Clontech). Filters were then washed 3 times for 30 minutes at 50° C.with 0.1×SSC, 0.1% SDS, autoradiographed overnight and compared tomanufacturer's grid patterns to identify positive clones.

B. Characterization of Zcytor19 Mouse Positive BACs.

Five zcytor19 mouse positive BAC clones from 129/SvJ Embryonic Stem Celllibraries (Release I and II) were obtained from Incyte Genomics. BACclones were grown within Escherichia coli host strain DH10B in liquidmedia and extracted using BAC large plasmid purification kit MKB-500(Incyte Genomics) according to manufacturer's instructions. 4 of 5 BACswere found to contain at least 2,000 bp of 5′ untranslated region,exon1, and exon 5 as determined by PCR. 100 ng of each BAC DNA was usedas template using the following conditions: PCR reactions were carriedout in 25 μl using 1.75 units of Advantage 2 polymerase (Clontech) inbuffer containing 67 mM Tris pH 8.8, 16.6 mM (NH₄)₂SO₄, 6.7 mM. MgCl₂, 5mM 2-Mercaptoethanol, 100 μg/ml gelatin, 10% Dimethyl Sulfoxide, 1 mMdeoxynucleotides, 140 nM forward and 140 nM reverse primer. PCRconditions were as follows 95° C. for 1 min; 30 cycles of 95° C. for 15seconds, 55° C. for 30 seconds, and 68° C. for 30 seconds; and 68° C.for 2 minutes; followed by a 4° C. hold. PCR products were analyzed byagarose gel electrophoresis. Using forward primer ZC40784 (SEQ ID NO:39)and reverse primer ZC40785 (SEQ ID NO:40) partial 5′ UTR was amplifiedand found to be 957 bp. Using forward primer ZC40786 (SEQ ID NO:41) andreverse primer ZC40787 (SEQ ID NO:42) partial 5′ UTR, complete exon 1and partial intron 1 was amplified and found to be approximately 950 bp.Using forward primer ZC39128 (SEQ ID NO:37) and forward primer ZC39129(SEQ ID NO:38) containing partial exon 6, complete intron 6 and partialexon 7 sequence was amplified and found to be 1,149 bp.

Four of the 5 BAC clones were found to contain at least 3,796 bp of 5′UTR and at 6,922 bp of 3′ UTR by Southern Blot analysis.Oligonucleotides ZC40784 (SEQ ID NO:39) and ZC39129 (SEQ ID NO:38) wereend labeled using T4 polynucleotide kinase (Roche) and used to probeSouthern Blots containing 5 BAC candidates digested with restrictionendonucleases EcoRI (Life Technologies) and XbaI (New England Biolabs).Results indicated 4 of 5 BACs contained at least 3,796 bp of 5′ UTR and5 of 5 BACs contained at least 6,922 bp of 3′ UTR.

C. Determination of Zcytor19 Mouse Intron 6 Sequence.

Oligonucleotides were designed to generate a PCR fragment containingpartial exon 6, complete intron 6 and partial exon 7 sequences.

PCR reactions were carried out in 25 μl using 1.75 units of Advantage 2polymerase (Clontech). 100 ng of 129/Sv mouse genomic DNA was used astemplate in buffer containing 67 mM Tris pH 8.8, 16.6 mM (NH₄)₂SO₄, 6.7mM MgCl₂, 5 mM 2-Mercaptoethanol, 100 μg/ml gelatin, 10% DimethylSulfoxide, 1 mM deoxynucleotides, 140 nM forward primer ZC39128 (SEQ IDNO:37) and 140 nM reverse primer ZC39129 (SEQ ID NO:38). PCR conditionswere as described above. PCR products were analyzed by agarose gelelectrophoresis and found to be 1,149 bp. PCR products were thenpurified using Qiaquick (Qiagen) PCR purification kit. Determination ofintron 6 sequence was made by sequence analysis using oligos ZC39128(SEQ ID NO:37) and ZC 39129 (SEQ ID NO:38).

D. Determination of Zcytor19 Mouse Intron 5 Sequence

Oligonucleotides were designed to generate a PCR fragment containingpartial exon5, complete intron5 and partial exon6. PCR reactions werecarried out in 25 μl using 1.75 units of Advantage 2 polymerase(Clontech). 100 ng of 129/Sv mouse genomic DNA was used as template inbuffer containing 67 mM Tris pH 8.8, 16.6 mM (NH₄)₂SO₄, 6.7 mM MgCl₂, 5mM 2-Mercaptoethanol, 100 μg/ml gelatin, 10% Dimethyl Sulfoxide, 1 mMdeoxynucleotides, 140 nM forward primer ZC39408 (SEQ ID NO:43) and 140nM reverse primer ZC39409 (SEQ ID NO:44). PCR conditions were as follows95° C. for 1 min; 30 cycles of 95° C. for 15 seconds, 55° C. for 30seconds, and 68° C. for 30 seconds; and 68° C. for 2 minutes; followedby a 4° C. hold. PCR products were analyzed by agarose gelelectrophoresis and found to be 356 bp. PCR products were then purifiedusing Qiaquick (Qiagen) PCR purification kit. Determination of intron 6sequence was made by sequence analysis using oligos ZC39408 (SEQ IDNO:43) and ZC 39409 (SEQ ID NO:44).

E. Design of Oligonucleotides for Generating of KO Constructs of theMouse Zcytor19 Gene

To investigate biological function of zytor19 gene, a knockout mousemodel is being generated by homologous recombination technology inembryonic stem (ES) cells. In this model, the coding exon 1, 2 and 3 aredeleted to create a null mutation of the zcytor19 gene. This deletionremoves the translation initiation codon, the signal domain and part ofthe extracellular domain of the zcytor19 protein, thus inactivating thezcytor19 gene.

ET cloning technique will be used to generate the KO vector (Stewart etal, Nucl. Acids Res. 27:6, 1999) First, Kanomycin resistance cassette isused to replace introns1, 2 and 3 of zcytor19 mouse gene. A forwardknockout oligonucleotide (SEQ ID NO:45) was designed to be 121nucleotides in length, having 52 bp of homology to the 5′UTR ofzcytor19m a 42 bp linker having SfiI, FseI, BamHI and HindIIIrestriction sites and 27 bp of homology to the 5′ end of the Kanomycinresistance cassette. A reverse knockout oligonucleotide (SEQ ID NO:46)was designed to be 1.25 nucleotides in length, having 50 bp of homologyto intron 3 of zcytor19 mouse, a 48 bp linker having SfiI, AscI, BamHIand HindIII restriction sites and 27 bp of homology to the 3′ end of theKanomycin resistance cassette. The above oligonucleotides can be used tosynthesize a PCR fragment 1073 bp in length containing the entireKanomycin resistance cassette with the first 52 bp having homology tothe 5′ UTR of zcytor19 mouse and the last 50 bp having homology tointron 3.

The fragment will then be used to construct a Knockout vector through ETCloning, in which cytor19 mouse positive BAC cell hosts are madecompetent through treatment with glycerol then transfected with theplasmid pBADalpha/beta/gamma(Amp). Resistance to chloramphenical andampicillin selects for transformed cell. Cells are then re-transformedwith the Kanomycin PCR fragment containing homology arms. The Beta andgamma recombination proteins of pBADalpha/beta/gamma(Amp) are induced bythe addition of arabinose to the growth media through the activation ofthe Red alpha gene. Recombinant BACs are selected for by resistance tokanomycin and ampicillin then screened by PCR. Once a recombinant BAC isidentified a fragment is subcloned containing at least 1,800 bp ofsequence upstream of kanomycin resistance cassette insertion and atleast 6,000 bp of sequence downstream into a pGEM7 derived vector. TheKanomycin resistance cassette is then replaced by standard ligationcloning with a IRES/LacZ/Neo-MC1 cassette. The IRES is an internalribosome entry sequence derived from encephalomyocarditis virus. It isfused in-frame to the reporter lacZ gene, linked to a polyA signal.Downstream of the IRES/LacZ reporter gene, MC1 promoter drives theexpression of a G418 resistance selectable marker Neo gene. Theselectable maker cassette contains termination codons in all threereading frames. Thus, the drug resistance gene Neo is used for selectionof homologous recombination events in embryonic stein (ES) cells.IRES/LacZ reporter gene will be used to monitor the expression of thereplaced gene after homologous recombination Homologous recombination ofthe knockout vector and the target locus in ES cells leads to thereplacement of a total 17,980 bp, including complete exons 1, 2 and 3,of the wild type locus with the IRES/LacZ/Neo-MC1 cassette, which isabout 5,200 bp in length.

F. Generation of zCytor19 KO Mice

The KO vector, described above, is linearized by PmeI digestion, andelectroporated into ES cells. Homologous recombination events areidentified by PCR screening strategy, and confirmed by Southern BlotAnalysis, using a standard KO protocol. See, A. L. Joyner, GeneTargeting. A Practical Approach. IRL Press 1993.

Once homologous recombination events are identified, ES cells will beexpanded, and injected into blastocysts to generate chimeras. Chimericmales will be used to breed to C57black females to achieve germ linetransmission of the null mutation, according to standard procedures. SeeHogan, B. et al., Manipulating the Mouse Embryo. A Laboratory Manual,Cold Spring Harbor Laboratory Press, 1994.

Heterozygous KO animals will be bred to test biological functions of thezcytor19 gene. Of offspring produced, ¼ should be wild type, ½ should beheterozygous, and ¼ should be homozygous. Homozygous will be analyzed indetails as described below.

G. Microscopic Evaluation of Tissues from Zcytor19 Homozygous Animals.

Since zcytor19 is expressed in following tissues, we will examine thesetissues carefully: colon, ovary placenta, pituitary, lymph node, smallintestine, salivary gland, rectum, prostate, testis, brain, lung,kidney, thyroid, spinal cord, bone marrow, and cervix.

Spleen, thymus, and mesenteric lymph nodes are collected and preparedfor histologic examination from transgenic animals expressing zcytor19.Other tissues which are routinely harvested included the following:Liver, heart, lung, spleen, thymus, mesenteric lymph nodes, kidney,skin, mammary gland, pancreas, stomach, small and large intestine,brain, salivary gland, trachea, esophagus, adrenal, pituitary,reproductive tract, accessory male sex glands, skeletal muscle includingperipheral nerve, and femur with bone marrow. The tissues are harvestedfrom homozygous animals as well as wild type controls. Samples are fixedin 10% buffered formalin, routinely processed, embedded in paraffin,sectioned at 5 microns, and stained with hematoxylin and eosin. Theslides are examined for histological, and pathological changes, such asinflammatory reactions, and hypo-proliferation of certain cell types.

H. Flow Cytometric Analysis of Tissues from Homozygous Mouse MutantsMissing Zcytor19.

Homozygous animals missing zcytor19 gene are to be sacrificed for flowcytometric analysis of peripheral blood, thymus, lymph node, bonemarrow, and spleen.

Cell suspensions are made from spleen, thymus and lymph nodes by teasingthe organ apart with forceps in ice cold culture media (500 ml RPMI 1640Medium (JRH Biosciences. Lenexa, Kans.); 5 ml 100×L-glutamine (GibcoBRL. Grand Island, N.Y.); 5 ml 100×Na Pyruvate (Gibco BRL); 5 ml 100×Penicillin, Streptomycin, Neomycin (PSN) (Gibco BRL) and then gentlypressing the cells through a cell strainer (Falcon, VWR Seattle, Wash.).Peripheral blood (200 ml) is collected in heparinized tubes and dilutedto 10 mls with HBSS containing 10 U Heparin/ml. Erythrocytes are removedfrom spleen and peripheral blood preparations by hypotonic lysis. Bonemarrow cell suspensions are made by flushing marrow from femurs withice-cold culture media. Cells are counted and tested for viability usingTrypan Blue (GIBCO BRL, Gaithersburg, Md.). Cells are resuspended in icecold staining media (HBSS, 1% fetal bovine serum, 0.1% sodium azide) ata concentration of ten million per milliliter. Blocking of Fe receptorand non-specific binding of antibodies to the cells was achieved byadding 10% normal goat sera and Fc Block (PharMingen, La Jolla, Calif.)to the cell suspension.

Cell suspensions are mixed with equal volumes of fluorochrome labeledmonoclonal antibodies (PharMingen), incubated on ice for 60 minutes andthen washed twice with ice cold wash buffer (PBS, 1% fetal bovine serum,0.1% sodium azide) prior to resuspending in 400 ml wash buffercontaining 1 mg/ml 7-AAD (Molecular Probes, Eugene, Oreg.) as aviability marker in some samples. Flow data was acquired on aFACSCalibur flow cytometer (BD Immunocytometry Systems, San Jose,Calif.). Both acquisition and analysis were performed using CellQuestsoftware (BD Immunocytometry Systems).

The cell populations in all lymphoid organs will be analyzed to detectabnormalities in specific lineages of T cell, B cell, or otherlymphocytes, and cellularity in these organs.

Example 14 Identification of Cells Expressing Zcytor19 Using In SituHybridization

Specific human tissues were isolated and screened for zcytor19expression by in situ hybridization. Various human tissues prepared,sectioned and subjected to in situ hybridization included normal andcarcinoma colon, cervical carcinoma, endometrial carcinoma, normal andcarcinoma ovary, normal and neoplasmic skin, fetal liver, lung, heartand MFH (muscle sarcoma). The tissues were fixed in 10% bufferedformalin and blocked in paraffin using standard techniques. Tissues weresectioned at 4 to 8 microns. Tissues were prepared using a standardprotocol. Briefly, tissue sections were deparaffinized with Histo-Clear®(National Diagnostics, Atlanta, Ga.) and then dehydrated with ethanol.Next they were digested with Proteinase K (50 μg/ml) (BoehringerDiagnostics, Indianapolis, Ind.) at 37° C. for 2 to 7 minutes. This stepwas followed by acetylation and re-hydration of the tissues.

One in situ probe was designed against the human zcytor19 (variant x1)sequence (INC7128744, as shown in SEQ ID NO: 25), containing the 3′UTRof zcytor19 using standard methods. T7 RNA polymerase was used togenerate an antisense probe. The probe was labeled using an In Vitrotranscription System (Riboprobe® in vitro Transcription System, Promega,Madison, Wis.) as per manufacturer's instruction, except that the probesdigoxigenin was used instead of radiolabeled rCTP and that the water wasadjusted to accommodate the reduced volume of the rNTP's. In situhybridization was performed with a digoxigenin-labeled zcytor19 probe(above). The probe was added to the slides at a concentration of 1 to 5omol/ml for 12 to 16 hours at 60° C. Slides were subsequently washed in2×SSC and 0.1×SSC at 55° C. The signals were amplified using TSA™(Tyramide Signal Amplification; PerkinElmer Life Sciences Inc., Boston,Mass.) and visualized with VECTOR Red substrate kit (VectorLaboratories, Burlingame, Calif.) as per manufacturer's instructions.The slides were then counter-stained with hematoxylin.

Signals were observed in several tissues tested: In colon carcinomatissues, weak signal was observed in carcinoma cells and a few immuneinfiltrations. However, there was no positive signal observed in thenormal colon and intestine, including cells in lamina propria,epithelium, immune nodules and peripheral ganglia nerve cells. Incervical carcinoma tissues, there is weak signal in carcinoma cells andsome cells in the immune nodules. In endometrial carcinoma tissues, weaksignals present in the carcinoma cells. In normal uterus tissues, nopositive signal was observed. In ovarian carcinoma samples, somecarcinoma cells are weakly positive. In normal ovary samples, someendothelium of capillaries and epithelium of large follicles may beweakly positive. In the skin carcinoma sample, the cancerous granularepithelium is strongly positive, while no positive signal is observed inthe normal skin. In fetal liver, signal is observed in a mixedpopulation of mononuclear cells in sinusoid spaces. In lung, zcytor19appears to be positive in type II alveolar epithelium. Occasionallybronchial epithelium may also be weakly positive. Macrophage-likemononuclear cells in the interstitial tissue are also positive. Inheart, myocytes are negative while some circulating mononuclear cellsare positive for zcytor19. In one of the samples, endothelium of thevessels may be weakly positive. Other tissues tested including a MFH(muscle sarcoma) sample and a Kaposi's sarcoma skin sample. There is noconclusive positive signal in these tissues.

Human tissues from cervical carcinoma, normal and carcinoma colon,duodenum, endometrial carcinoma, normal and carcinoma ovary, uterus,heart, liver, lung, muscle sarcoma, and normal and carcinoma skin werescreened for zcytor19 expression by in situ hybridization. The tissueswere fixed in 10% buffered formalin and blocked in paraffin usingstandard techniques. Tissues were sectioned at 5 microns. Tissues wereprepared using a standard protocol. Briefly, tissue sections weredeparaffinized with HistoClear (National Diagnostics, Atlanta, Ga.) andthen dehydrated with ethanol. Next they were digested with Proteinase K(50 μg/ml) (Boehringer Diagnostics, Indianapolis, Ind.) at 23° C. for4-15 minutes. This step was followed by acetylation and re-hydration ofthe tissues.

One in situ probe was designed against the human zcytor19 sequence.Plasmid DNA 100933 was digested with restriction enzyme HindIII, whichcovers 0.7 kb from the end of 3′UTR. The T-7 RNA polymerase was used togenerate an antisense probe. The probe was labeled with digoxigenin(Boehringer) using an In Vitro transcription System (Promega, Madison,Wis.) as per manufacturer's instruction.

In situ hybridization was performed with a digoxigenin- orbiotin-labeled zcytor19 probe (above). The probe was added to the slidesat a concentration of 1 to 5 pmol/ml for 12 to 16 hours at 60° C. Slideswere subsequently washed in 2×SSC and 0.1×SSC at 55° C. The signals wereamplified using tyramide signal amplification (TSA) (TSA, in situindirect kit; NEN) and visualized with Vector Red substrate kit (VectorLab) as per manufacturer's instructions. The slides were thencounter-stained with hematoxylin (Vector Laboratories, Burlingame,Calif.).

Positive signal were observed in most of carcinoma samples. In cervicalcarcinoma, carcinoma epithelial cells were positive. There were alsosome signals in a subset of lymphocytes in the lymphoid follicles.Similarly, both carcinoma and some immune cells were positive in thecolon carcinoma samples, while normal colon samples were negative. Weakstaining was also in the endometrial carcinoma and ovarian carcinoma,while normal ovary and uterus were negative. There was weak staining inthe cancer area of the muscle sarcoma sample. Keratinocytes werepositive in the skin carcinoma and Kaposi's sarcoma samples, while nostaining was observed in the normal skin. In heart and liver, a subsetof cells possibly circulating WBC, were positive for zcytor19. Itappears endothelial cells in some vessels may also be positive. In lung,type II pneumocytes and macrophage-like cells were positive. Bronchialepithelium and endothelium were also positive in some lung specimens. Insummary, zcytor19 appears to be up-regulated in carcinoma cells. Thereis low level of zcytor19 mRNA in a subset of lymphocytes and endothelialcells.

Because zcytor19 is expressed in these specific tumor tissues, zcytor19polynucleotides, polypeptides and antibodies can be used as a tumormarker as disclosed herein. Moreover, an antibody to zcytor19 could haveanti-tumor activity, as well as toxin-conjugates, cytokine conjugates orother conjugates of an antibody, or the zcytor19 receptor ligand itself.The antagonist of zcytor19 ligand, such as anti-zcytor19 antibodies orsoluble receptors can also act as anti-tumor reagents.

Example 15 Construction of BaF3 Cells Expressing the Zcytor19 Receptor(BaF3 Zcytor19 Cells) with Puromycin Resistant and Zeomycin ResistantVectors

Two types of BaF3 cells expressing the full-length zcytor19 receptorwere constructed using 30 μg of zcytor19 expression vectors, oneresistant to puromycin, one resistant to zeomycin described below. TheBaF3 cells expressing the zcytor19 receptor mRNA with puromycinresistance were designated as BaF3/zcytor19-p. The BaF3 cells expressingthe zcytor19 receptor mRNA with zeomycin resistance were designated asBaF3/zcytor19-z

A. Construction of BaF3 Cells Expressing the Zcytor19 Receptor

BaF3, an interleukin-3 (IL-3) dependent pre-lymphoid cell line derivedfrom murine bone marrow (Palacios and Steinmetz, Cell 41: 727-734, 1985;Mathey-Prevot et al., Mol. Cell. Biol. 6: 4133-4135, 1986), wasmaintained in complete media (RPMI medium (JRH Bioscience Inc., Lenexa,Kans.) supplemented with 10% heat-inactivated fetal calf serum, 2 ng/mlmurine IL-3 (mIL-3) (R & D, Minneapolis, Minn.), 2 mM L-glutaMax-1™(Gibco BRL), 1 mM Sodium Pyruvate (Gibco BRL). Prior to electroporation,pZP-5N/CRF2-4 was prepared and purified using a Qiagen Maxi Prep kit(Qiagen) as per manufacturer's instructions. BaF3 cells forelectroporation were washed twice in PBS (Gibco BRL) and thenresuspended in RPMI media at a cell density of 10⁷ cells/ml. One ml ofresuspended BaF3 cells was mixed with 30 μg of the pZP-7p/zcytor19plasmid DNA, or 30 □g of the pZP-7z/zcytor19 plasmid DNA, andtransferred to separate disposable electroporation chambers (GIBCO BRL).The cells were given two serial shocks (800 1Fad/300 V.; 1180 1Fad/300V.) delivered by an electroporation apparatus (CELL-PORATOR™; GIBCOBRL), with a 1 minute rest between the shocks. After a 5 minute recoverytime, the electroporated cells were transferred to 50 ml of completemedia and placed in an incubator for 15-24 hours (37° C., 5% CO₂). Thecells were then spun down and resuspended in 50 ml of complete mediacontaining Puromycin (Clonetech) selection (2 □g/ml) for the cellstransfected with pZP-7p/zcytor19, or Zeocin selection (1:150-1:333) forthe cells transfected with pZP-7z/zcytor19, and placed in a T-162 flaskto isolate the antibiotic-resistant pools. Pools of the transfected BaF3cells, hereinafter called BaF3/zcytor19-puro and BaF3/zcytor19-zeocells, were assayed for expression of zcytor19 by RT-PCR.

B. Confirmation of Zcytor19 Expression by RT-PCR

The BaF3/zcytor19-puro and BaF3/zcytor19-zeo cells were harvested forRNA, which was then put into a reverse transcriptase reaction, andsubsequently tested by PCR for the presence of zcytor19.

Flasks of cells were grown to confluence, then 10 ml were removed andspun down to obtain a cell pellet. RNA was purified from the pelletusing the RNeasy Total RNA Purification kit, with the additionalRNase-free DNase set (Qiagen), following the manufacturer's protocol.Reverse transcription was then done on the samples using theStrataScript RT-PCR kit (Stratagene), following the manufacturer'sprotocol through the completion of the RT reaction. PCR was then done bymixing 0.2 pmol each of primers ZC40279 and ZC37863, 0.2 mM of dNTP mix(Roche) containing equal amounts of each nucleotide, 5 □l of 10× cDNAPCR Reaction Buffer (Clonetech), 3 □l DNA from the RT reaction, 0.5 □lAdvantage2 Polymerase (Clonetech), made to a final volume of 50 □l withwater. The reaction ran for 95° C., 5 min, then 30 cycles of 95° C. 30sec, 60° C. 30 sec, 72° C. 1 min, then 72° C. 7 min and a 4° C. soak, ona Perkin Elmer GeneAmp PCR System 2400. The samples were mixed with 3 mlloading dye, and 25 ml was run on a 1% OmniPur Agarose (Merck) gel.Zcytor19 bands were detected on the gel for both BaF3/zcytor19-puro andBaF3/zcytor19-zeo, indicating that those cells are expressing the gene.

Example 16 Polyclonal Antibodies

Polyclonal antibodies are prepared by immunizing 2 female New Zealandwhite rabbits with the purified recombinant protein huzcytor19/MBP-6H.The rabbits are each given an initial intraperitoneal (ip) injection of200 μg of purified protein in Complete Freund's Adjuvant followed bybooster ip injections of 100 μg peptide in Incomplete Freund's Adjuvantevery three weeks. Seven to ten days after the administration of thesecond booster injection (3 total injections), the animals are bled andthe serum is collected. The animals are then boosted and bled everythree weeks.

The huzcyotr19/MBP-6H specific rabbit serum is pre-adsorbed of anti-MBPantibodies using a CNBr-SEPHAROSE 4B protein column (Pharmacia LKB,Peapack, N.J.) that is prepared using 10 mg of purified recombinant MBPper gram of CNBr-SEPHAROSE. The huzcytor19-specific polyclonalantibodies are affinity purified from the rabbit serum using aCNBr-SEPHAROSE 4B protein column that is prepared using 10 mg of thespecific antigen purified recombinant protein huzcytor19/MBP-6H followedby 20× dialysis in PBS overnight. Huzcytor19-specific antibodies arecharacterized by ELISA using 500 ng/ml of the purified recombinantproteins huzcytor19/MBP-6H or huzcytor19-Fc4 as antibody targets. Thelower limit of detection (LLD) of the rabbit anti-huzcytor19/MBP-6Haffinity purified antibody on its specific purified recombinant antigenhuzcytor19/MBP-6H and on purified recombinant huzcytor19-Fc4 isdetermined.

Example 17 Signal Transduction Reporter Assay

A signal transduction reporter assay can be used to determine thefunctional interaction of zcyto20, zcyto21, zcyto22, zcyto24, andzcyto25 with zcytor19. Human embryonal kidney (HEK) cells aretransfected with a reporter plasmid containing an interferon-stimulatedresponse element (ISRE) driving transcription of a luciferase reportergene in the presence or absence of pZP7 expression vectors containingcDNAs for class II cytokine receptors (including human DIRS1, IFNaR1,IFNaR2 and Zcytor19 (SEQ ID NO:23)). Luciferase activity followingstimulation of transfected cells with class II ligands (includingzcyto20 (SEQ ID NO:52), zcyto21 (SEQ ID NO:55), zcyto22 (SEQ ID NO:57),zcyto10, huIL10 and huIFNa-2a) reflects the interaction of the ligandwith transfected and native cytokine receptors on the cell surface. Theresults and methods are described below.

Cell Transfections

293 HEK cells were transfected as follows: 700,000 293 cells/well (6well plates) were plated approximately 18 h prior to transfection in 2milliliters DMEM+10% fetal bovine serum. Per well, 1 microgrampISRE-Luciferase DNA (Stratagene), 1 microgram cytokine receptor DNA and1 microgram pIRES2-EGFP DNA (Clontech,) were added to 9 microlitersEugene 6 reagent (Roche Biochemicals) in a total of 100 microlitersDMEM. Two micrograms pIRES2-EGFP DNA was used when cytokine receptor DNAwas not included. This transfection mix was added 30 minutes later tothe pre-plated 293 cells. Twenty-four hours later the transfected cellswere removed from the plate using trypsin-EDTA and replated atapproximately 25,000 cells/well in 96 well microtiter plates.Approximately 18 h prior to ligand stimulation, media was changed toDMEM+0.5% FBS.

Signal Transduction Reporter Assays

The signal transduction reporter assays were done as follows: Followingan 18 h incubation at 37° C. in DMEM+0.5% FBS, transfected cells werestimulated with dilutions (in DMEM+0.5% FBS) of the following class IIligands; zcyto20, zcyto21, zcyto22, zcyto10, huIL10 and huIFNa-2a.Following a 4-hour incubation at 37° C., the cells were lysed, and therelative light units (RLU) were measured on a luminometer after additionof a luciferase substrate. The results obtained are shown as the foldinduction of the RLU of the experimental samples over the medium alonecontrol (RLU of experimental samples/RLU of medium alone=foldinduction). Table 14 shows that zcyto20, zcyto21 and zcyto22 induce ISREsignaling in 293 cells transfected with ISRE-luciferase giving a 15 to17-fold induction in luciferase activity over medium alone. The additionof zcytor19 DNA to the transfection mix results in a 6 to 8-fold furtherinduction in ISRE signaling by zcyto20, zcyto21 and zcyto22 giving a 104to 125-fold total induction. None of the other transfected class IIcytokine receptor DNAs resulted in increased ISRE signaling. Theseresults indicate that zcyto20, zcyto21 and zcyto22 functionally interactwith the zcytor19 cytokine receptor. Table 8 also shows that huIFNa-2acan induce ISRE signaling in ISRE-luciferase transfected 293 cellsgiving a 205-fold induction of luciferase activity compared to mediumalone. However, the addition of zcytor19 DNA to the transfection leadsto an 11-fold reduction in ISRE-signaling (compared to ISRE-luciferaseDNA alone), suggesting that zcytor19 over-expression negatively effectsinterferon signaling, in contrast to the positive effects of zcytor19over-expression on zcyto20, zcyto21 and zcyto22 signaling.

TABLE 8 Interferon Stimulated Response Element (ISRE) Signaling ofTransfected 293 Cells Following Class II Cytokine Stimulation (FoldInduction) Ligand ISRE-Luc. ISRE-Luc./Zcytor19 Zcyto20 (125 ng/ml) 15125 Zcyto21 (125 ng/ml) 17 108 Zcyto22 (125 ng/ml) 17 104 HuIFNa-2a (100ng/ml) 205 18 Zcyto10 (125 ng/ml) 1.3 1 HuIL10 (100 ng/ml) 1 0.5

Example 18 Identification of IL10Rb (CRF2-4) as a Receptor Subunit forZcytor19

A: IL10Rb Neutralizing Antibody Inhibits ISRE Signaling:

A signal transduction reporter assay was used to determine thefunctional interaction of zcyto20, zcyto21, and zcyto22 with zcytor19and IL10Rb (CRF2-4). Human embryonal kidney (HEK) cells or humanembryonal kidney (HEK) cells stably overexpressing human zcytoR19 weretransfected with a reporter plasmid containing an interferon-stimulatedresponse element (ISRE) driving transcription of a luciferase reporter.Luciferase activity following stimulation of transfected cells withclass II ligands (including zcyto20, zcyto21, zcyto22 and huIFNa-2a) inthe presence or absence of a neutralizing antibody to IL10Rb (CRF2-4)reflects the interaction of the ligand with cytokine receptors on thecell surface. The results and methods are described below.

Cell Transfections:

To produce 293 HEK cells stably overexpressing human zcytoR19, 293 cellswere transfected as follows: 300,000 293 cells/well (6 well plates) wereplated approximately 6 h prior to transfection in 2 milliliters DMEM+10%fetal bovine serum. Per well, 2 micrograms of a pZP7 expression vectorcontaining the cDNA of human zcytoR19 (SEQ ID NO:23) was added to 6microliters Fugene 6 reagent (Roche Biochemicals) in a total of 100microliters DMEM. This transfection mix was added 30 minutes later tothe pre-plated 293 cells. Forty-eight hours later the transfected cellswere placed under 2 microgram/milliliter puromicin selection. Puromicinresistant cells were carried as a population of cells.

The 293 HEK cells (wild type or overexpressing human zcytoR19) weretransfected as follows: 700,000 293 cells/well (6 well plates) wereplated approximately 18 h prior to transfection in 2 millilitersDMEM+10% fetal bovine serum. Per well, 1 microgram pISRE-Luciferase DNA(Stratagene) and 1 microgram pIRES2-EGFP DNA (Clontech) were added to 6microliters Fugene 6 reagent (Roche Biochemicals) in a total of 100microliters DMEM. This transfection mix was added 30 minutes later tothe pre-plated 293 cells. Twenty-four hours later the transfected cellswere removed from the plate using trypsin-EDTA and replated atapproximately 25,000 cells/well in 96 well microtiter plates.Approximately 18 h prior to ligand stimulation, media was changed toDMEM+0.5% FBS.

Signal Transduction Reporter Assays:

The signal transduction reporter assays were done as follows: Followingan 18 h incubation at 37 degrees in DMEM+0.5% FBS, transfected cellswere pretreated with a neutralizing polyclonal goat antibody to IL10Rb(2.5 micrograms/ml for zcyto21; 8 micrograms/ml for zcyto20 and zcyto22,R&D Systems) or PBS for 1 hour at 37 C. Human embryonal kidney (HEK)cells stably overexpressing human zcytoR19 were also pretreated with anon-neutralizing polyclonal goat antibody to IFNAR1 (8 micrograms/ml,R&D Systems) as an antibody control for experiments involving zcyto20and zcyto22. Pretreated cells were stimulated with dilutions (inDMEM+0.5% FBS) of the following class II ligands; zcyto20, zcyto21, orzcyto22. As a control, huIFNa-2a was run in each experiment. Following a4-hour incubation at 37 degrees, the cells were lysed, and the relativelight units (RLU) were measured on a luminometer after addition of aluciferase substrate. The results obtained are shown as the foldinduction of the RLU of the experimental samples over the medium alonecontrol (RLU of experimental samples/RLU of medium alone=foldinduction).

Tables 9 and 10 show that induction of ISRE signaling by zcyto20 isinhibited by pretreatment of wild type 293 cells or 293 cellsoverexpressing human zcytoR19 with a neutralizing antibody to IL10Rb. Noor little inhibition is seen of huIFNa-2a induction of ISRE signaling.These results indicate that zcyto20 requires interaction with IL10Rb(CRF2-4) for maximal induction of ISRE signaling and that the receptorfor zcyto20 is the heterdimeric combination of zcytoR19 and IL10Rb(CRF2-4).

TABLE 9 IL10Rb Inhibition of ISRE Signaling of Transfected wild-type 293Cells Following Class II Cytokine Stimulation (Fold Induction) Zcyto20 +HuIFNa-2a + Cytokine IL10Rb IL10Rb Concentration neutralizingneutralizing (ng/ml) Zcyto20 Antibody HuIFNa-2a Antibody 100 8.4 0.8 152102 10 4 0.9 160 117 1 1 0.9 90 69 0.1 1 1 12 6 0.01 1 0.8 1.2 1 0 1 1 11

TABLE 10 IL10Rb Inhibition of ISRE Signaling of TransfectedzcytoR19-overexpressing 293 Cells Following Class II CytokineStimulation (Fold Induction) Zcyto20 + HuIFNa-2a + Cytokine IL10RbIL10Rb Concentration neutralizing neutralizing (ng/ml) Zcyto20 AntibodyHuIFNa-2a Antibody 100 91 60 16 16 10 97 23 14 15 1 68 1.3 8 8.4 0.1 61.1 1.5 1.9 0.01 1.1 1.2 1.2 1.3 0 1 1 1 1

Tables 11 and 12 show that ISRE signaling by zcyto21 is inhibited bypretreatment of wild type 293 cells or 293 cells overexpressing humanzcytoR19 with a neutralizing antibody to IL10Rb. No inhibition is seenof huIFNa-2a induction of ISRE signaling. These results indicate thatzcyto21 requires interaction with IL10Rb (CRF2-4) for maximal inductionof ISRE signaling and that the receptor for zcyto21 is the heterdimericcombination of zcytoR19 and IL10Rb (CRF2-4).

TABLE 11 IL10Rb Inhibition of ISRE Signaling of Transfected wild-type293 Cells Following Class II Cytokine Stimulation (Fold Induction)Zcyto21 + HuIFNa-2a + Cytokine IL10Rb IL10Rb Concentration neutralizingneutralizing (ng/ml) Zcyto21 Antibody HuIFNa-2a Antibody 100 4.1 1.8 3130 10 3.2 1.4 32 31 1 1.5 1.3 16.3 15 0.1 1.1 1.3 1.4 2 0.01 1.2 1.3 1.11.2 0.001 1.2 1.3 0.9 2.1 0 1 1 1 1

TABLE 12 IL10Rb Inhibition of ISR) Signaling of TransfectedzcytoR19-overexpressing 293 Cells Following Class II CytokineStimulation (Fold Induction) Zcyto21 + HuIFNa-2a + Cytokine IL10RbIL10Rb Concentration neutralizing neutralizing (ng/ml) Zcyto21 AntibodyHuIFNa-2a Antibody 100 45 31 9 7.7 10 48 28 9 8.5 1 35 5.8 4.3 4.3 0.13.5 1 1.4 1.3 0.01 1.5 1.1 0.9 1.2 0.001 1.1 1 1.2 1 0 1 1 1 1

Tables 13 and 14 show that induction of ISRE signaling by zcyto22 isinhibited by pretreatment of wild type 293 cells or 293 cellsoverexpressing human zcytoR19 with a neutralizing antibody to IL10Rb. Noor little inhibition is seen of huIFNa-2a induction of ISRE signaling.These results indicate that zcyto22 requires interaction with IL10Rb(CRF2-4) for maximal induction of ISRE signaling and that the receptorfor zcyto22 is the heterdimeric combination of zcytoR19 and IL10Rb(CRF2-4).

TABLE 13 IL10Rb Inhibition of ISRE Signaling of Transfected wild-type293 Cells Following Class II Cytokine Stimulation (Fold Induction)Zcyto22 + HuIFNa-2a + Cytokine IL10Rb IL10Rb Concentration neutralizingneutralizing (ng/ml) Zcyto22 Antibody HuIFNa-2a Antibody 100 11 1.2 152102 10 8 1 160 117 1 1.8 0.8 90 69 0.1 1.2 0.8 12 6 0.01 0.9 0.9 1.2 1 01 1 1 1

TABLE 14 IL10Rb Inhibition of ISRE Signaling of TransfectedzcytoR19-overexpressing 293 Cells Following Class II CytokineStimulation (Fold Induction) Zcyto22 + HuIFNa-2a + Cytokine IL10RbIL10Rb Concentration neutralizing neutralizing (ng/ml) Zcyto22 AntibodyHuIFNa-2a Antibody 100 82 76 16 16 10 97 39 14 15 1 69 2.3 8 8.4 0.1 8.41.1 1.5 1.9 0.01 1 1.3 1.2 1.3 0 1 1 1 1

B: A: Anti-IL10Rb Antibody Blocks Antiviral Activity

An antiviral assay was performed to determine the ability of anti-IL10Rbantibody to block the antiviral activity of zcyto20. The assay wascarried out using 293 HEK cells (wild type or overexpressing humanzcytoR19). On the first day, antibodies (anti-human IL10R beta,anti-human Leptin receptor, R&D Systems) were diluted into cell media at5 micrograms/ml and then plated with 50,000 cells per well into a96-well plate. Following a one-hour incubation at 37° C., zcyto20-CEE(from example 3) (200 ng/ml for wild-type 293 cells, 0.5 ng/ml for 293cells overexpressing human zcytoR19) or human interferon-a-2a (1 ng/mlfor wild-type 293 cells, 100 ng/ml for 293 cells overexpressing humanzcytoR19) were added to the wells and incubated overnight at 37° C. Thenext day, the medium was removed and replaced with medium containingencephalomyocarditis virus (EMCV) at a multiplicity of infection of 0.1.The cells were then incubated at 37° C. overnight. Subsequently, 25 uLof 5 mg/ml Methylthiazoletetrazolium (MTT)(Sigma) were added to eachwell, incubated 2 hours at 37 degrees, and wells were then extractedwith 100 uL extraction buffer (12.5% SDS, 45% DMF). Following overnightincubation at 37° C., the optical density at 570 nM was measured on aSpectromax plate reader (Molecular Devices, CA). Decreased opticaldensity (570 nm) indicates decreased cell survival (loss of antiviralactivity). The optical densities (570 nm) for the different experimentalconditions are shown in Table 15 below. The results indicate thatblocking human IL10 receptor beta specifically neutralizes the antiviralactivity of zcyto20 without effecting interferon-a-2a activity. Thisindicates that human IL10 receptor beta is part of the receptor complex(including human zcytoR19) involved in zcyto20 antiviral activity.

TABLE 15 Optical Density (570 nm) of ECMV-Infected Cytokine-TreatedCells HuzcytoR19- HuzcytoR19- Wild-type 293 Wild-type 293 Cells:overexpressing 293 overexpressing 293 Cytokine Cells: Anti-IL10RbAnti-LeptinR Cells: Anti-IL10Rb Cells: Anti-LeptinR Zcyto20-CEE 0.941.88 0.95 2.24 HuIFNa-2a 2.58 2.4 2.18 2.05

C: Zcyto20, Zcyto21, and Zcyto22 Signaling is Enhanced by Coexpressionof zcytor19 and IL10Rb:

A signal transduction reporter assay was used to determine thefunctional interaction of zcyto20, zcyto21 and zcyto22 with zcytor19 andIL10Rb (CRF2-4). Hamster kidney (BHK) cells were transfected with areporter plasmid containing an interferon-stimulated response element(ISRE) driving transcription of a luciferase reporter gene in thepresence or absence of pZP7 expression vectors containing cDNAs forclass II cytokine receptors Zcytor19 and IL10Rb (CRF2-4). Luciferaseactivity following stimulation of transfected cells with class IIligands (including zcyto20, zcyto21 and zcyto22) reflects theinteraction of the ligand with transfected and native cytokine receptorson the cell surface. The results and methods are described below.

Cell Transfections

BHK-570 cells were transfected as follows: 200,000 BHK cells/well (6well plates) were plated approximately 5 h prior to transfection in 2milliliters DMEM+5% fetal bovine serum. Per well, 1 microgrampISRE-Luciferase DNA (Stratagene), 1 microgram cytokine receptor DNA and1 microgram pIRES2-EGFP DNA (Clontech) were added to 9 microlitersFugene 6 reagent (Roche Biochemicals) in a total of 100 microlitersDMEM. Two micrograms pIRES2-EGFP DNA was used when cytokine receptor DNAwas not included. This transfection mix was added 30 minutes later tothe pre-plated BHK cells. Twenty-four hours later the transfected cellswere removed from the plate using trypsin-EDTA and replated atapproximately 25,000 cells/well in 96 well microtiter plates.Approximately 18 h prior to ligand stimulation, media was changed toDMEM+0.5% FBS.

Signal Transduction Reporter Assays

The signal transduction reporter assays were done as follows: Followingan 18 h incubation at 37° C. in DMEM+0.5% FBS, transfected cells werestimulated with dilutions (in DMEM+0.5% FBS) of zcyto20, zcyto21,zcyto22, zcyto24, and zcyto25 ligands. Following a 4-hour incubation at37 degrees, the cells were lysed, and the relative light units (RLU)were measured on a luminometer after addition of a luciferase substrate.The results obtained are shown as the fold induction of the RLU of theexperimental samples over the medium alone control (RLU of experimentalsamples/RLU of medium alone=fold induction). Table 16 shows thatzcyto20, zcyto21 and zcyto22 induce ISRE signaling in BHK cellstransfected with ISRE-luciferase and zcytoR19 in a dose-dependentmanner. The addition of IL10Rb (CRF2-4) DNA to the transfection mixresults in a half-maximal induction of signaling at a 10-100 fold lowercytokine dose. No response was seen with ISRE transfection alone. Theseresults show that the ability of zcyto20, zcyto21 and zcyto22 to signalthrough the interferon stimulated response element is enhanced bycoexpression of zcytoR19 and IL10Rb (CRF2-4) indicating that thereceptor for zcyto20, zcyto21 and zcyto22 is the heterdimericcombination of zcytoR19 and IL10Rb (CRF2-4).

TABLE 16 Interferon Stimulated Response Element (ISRE) Signaling ofTransfected BHK Cells Following Class II Cytokine Stimulation (FoldInduction) zcyto21/ zcyto22/ zcyto21/ cells zcyto22/ cells zcyto20/cellscells transfected cells transfected Class II transfected transfectedwith transfected with Ligand zcyto20/cells with zcytoR19 with zcytoR19with zcytoR19 Conc. transfected with and IL10Rb zcytoR19 and IL10RbzcytoR19 and IL10Rb (ng/ml) zcytoR19 alone (CRF2-4) alone (CRF2-4) alone(CRF2-4) 1000 2.25 2.1 3.3 2.2 1.8 2.2 100 2.2 2.6 2.6 2.5 2 2.2 10 2.12.4 2.4 2.6 1.9 2.7 1 1.3 2.5 2 2.5 1.5 2.7 0.1 1.25 2.1 1.4 2.2 1.1 2.40.01 1.2 1.6 1.4 1.6 1.2 1.7 0.001 1.4 1.5 1.3 1.3 1.2 1.3 0 1 1 1 1 1 1

Example 19 Binding of Ligands to Soluble Receptors

The binding of the ligands (zcyto20, zcyto21, zcyto22, zcyto24, andzcyto25) to soluble receptors can be assayed using an iodo-bead labelingmethod. For example, ¹²⁵I labeled zcyto21-CEE is labeled (1.2×10⁷CPM/ml; 1.5 ng/ul; and 8.6×10⁶ CPM/ug).

Fifty nanograms of the ¹²⁵I labeled zcyto21-cEE (See Example 3) (399,600CPM) is combined with 1000 ng of cold zcytor19/Fc4 homodimer receptor,1000 ng cold zcytor19/CRF2-4 heterodimer receptor, or 1000 ng of acontrol Class II cytokine receptor/Fc4 receptor as a control with about10,000 ng of cold zcyto21 as a competitor. Samples are incubated for 2hours at 4° C., after which 30 ul protein-G (Zymed San Francisco,Calif.) is added to each sample. Samples are incubated for 1 hour at 4°C., and washed 3 times with PBS. Radioactivity of the washed protein-Gis measured in a gamma counter (Packard Instruments, Downers Grove,Ill.).

Example 20 Flow Cytometry Staining of Human Monocytes with Zcyto20 andZcyto21-Biotin

Peripheral blood leukocytes (PBLs) were isolated by Ficoll Hypaque(Amersham, Sweden) separation from heparinized human blood. The PBLswere cultured at 37° C. in standard media at a density of 1×10^(e)6cells per milliliter in 6-well tissue culture plates. Followingovernight incubation, the PBLs were harvested and stained withbiotinylated zcyto20-cee and zcyto21-cee (See Example 18) at aconcentration of 10 ug/ml. Staining was detected withPhycoerythrin-labeled streptavidin (Pharmingen, CA, USA) that wasprepared at a dilution of 1:1000. Following staining the PBLs were fixedin 2% Paraformaldehyde, and read on a Facscaliber (Becton Dickinson, SanDiego, Calif.). The data was analyzed using Cellquest software (BectonDickinson). Results indicate that both biotinylated zcyto20-cee andzcyto21-cee stain cells in the myeloid gate of peripheral bloodleukocytes. Cells in the lymphoid gate do not bind zcyto20-cee andzcyto21-cee.

Example 21 Expression of Zcytor19 by Northern Analysis

Northern blots were probed to determine the tissue distribution ofzcytor19. A human zcytor19 cDNA fragment was obtained using PCR withgene specific primers, 5′ ZC40285 as shown in SEQ ID NO: 21; and 3′ ZC40286, as shown in SEQ ID NO: 22. The template was cloned human zcytor19cDNA. (SEQ ID NO: 23) The PCR fragment was gel purified, and ˜25 ng waslabeled with P³² α-dCTP using the Prime-It® RmT random prime labelingkit (Stratagene, LaJolla, Calif.).

The following Northern blots (Clontech, Palo Alto, Calif.) were probedfor mRNA expression of zcytor19: (1) a human cancer cell line blot C,which contains RNA samples from each of the following cancer cell lines:promyelocytic leukemia HL-60, HELA S3, chronic myelogenous leukemiak-562, lymphoblastic leukemia MOLT-4, Burkitt's lymphoma RAJI,colorectal adenocarcinoma SW480, lung carcinoma A549, and melanomaG-361; (2) a human MTN H blot, which contains mRNA from the followingtissues: heart, whole brain, placenta, lung, liver, skeletal muscle,kidney, and pancreas; (3) a human MTN H3 which contains mRNA from thefollowing tissues: stomach, thyroid, spinal cord, lymph node, trachea,adrenal gland, and bone marrow; and (4) a human MTN H4, which containsmRNA from the following tissues: spleen, thymus, prostate testis,uterus, small intestine, colon, and peripheral blood leukocytes.Hybridizations were all performed in ULTRAhyb™ UltrasensitiveHybridization Buffer (Ambion, Austin, Tex.) according the manufacturer'srecommendations, which the exception that an additional 0.2 mg/ml salmonsperm DNA was added to the hybridization and prehybridization buffers tolower non-specific hybridization. Following hybridization, non-specificradioactive signal was removed by treating the blots with 0.1×SSC/0.5%SDS at 50° C. The blots were exposed using BioMax MR Film andintensifying screens (Eastman Kodak, Rochester, N.Y.), per themanufacturer's recommendations for 3 days.

Expression of a ˜4.5 kb transcript was in greatest in heart, skeletalmuscle, pancreas and prostate tissue, in addition to in the Burkitt'slymphoma (RAJI) cell line. Lower levels were seen in multiple othertissues. In addition, there was an ˜2 kb transcript which was generallyless abundant than the larger transcript, but also present in many ofthe tissues and cell lines. Testis tissue, in addition to having the 2and 4.5 kb transcripts, may also have ˜4 kb and 1.4 kb transcripts.Adrenal gland demonstrated equal levels of expression of the 4.5 kb and2 kb transcripts.

Example 22 Human Zcytor19 Expression Based on RT-PCR Analysis ofStimulated Versus Non-Stimulated Cells

Gene expression of zcytor19 was examined using RT-PCR analysis of thefollowing cell types: Hela, 293, Daudi, CD14+, U937, and HL-60.

First-strand cDNA synthesis from total RNA was carried out using acommercially available first-strand synthesis system for RT-PCR(Invitrogen life technologies, Carlsbad, Calif.). The subsequent PCRreactions were set up using zcytor19×1 (SEQ ID NO:1) and zcytor19×2 (SEQID NO:18) specific oligo primers ZC40288 (SEQ ID NO:65) and ZC40291 (SEQID NO:66) which yield a 806 bp and 892 bp product, respectively, QiagenHotStarTaq DNA Polymerase and Buffer, (Qiagen, Inc., Valencia, Calif.),GeneAmp dNTPs (Applied Biosystems, Foster City, Calif.), RediLoad™ dye(Research Genetics, Inc., Huntville, Ala.) and 2 μl first-strand cDNA(10% of the first-strand reaction) from the respective cell types. ThePCR cycler conditions were as follows: an initial 1 cycle 15 minutedenaturation at 95° C., 35 cycles of a 45 second denaturation at 94° C.,1 minute annealing at 63° C. and 1 minute and 15 second extension at 72°C., followed by a final 1 cycle extension of 7 minutes at 72° C. Thereactions were separated by electrophoresis on a 2% agarose gel (EMScience, Gibbstown, N.J.) and visualized by staining with ethidiumbromide.

Bands of the correct size were seen in Hela±IFN-beta (only the 892 bpband), 293+ Parental Adv, Daudi±IFN-beta, Daudi±TN-alpha, CD14+activated, HL-60 activated. No band was observed in CD14+ resting, U937resting and activated, and HL-60 resting. These results show inductionof zcytoR19 expression upon activation or differentiation of monocytesor monocyte cell lines.

Example 23 Construct for Generating hzcytor19/hCRF2-4 Heterodimer

A cell line expressing a secreted hzcytor19/hCRF2-4 heterodimer wasconstructed. In this construct, the extracellular domain of hzcytor19was fused to the heavy chain of IgG gamma1 (Fc4) (SEQ ID NO:14 and SEQID NO:15) with a Glu-Glu tag (SEQ ID NO:11) at the c-terminus, while theextracellular domain of CRF2-4 (SEQ ID NO:64) was fused to Fc4 with aHis tag at the C-terminus. For both of the hzcytor19 and hCRF2-4 arms ofthe heterodimer, a Gly-Ser spacer of 12 amino acids was engineeredbetween the extracellular portion of the receptor and the n-terminus ofFc4. In addition, a thrombin cleavage site was engineered between theFc4 domain and the c-terminal tag to enable possible proteolytic removalof the tag.

For construction of the hzcytor19/Fc4-CEE portion of the heterodimer,the extracellular portion of hzcytor19 was amplified by PCR from a braincDNA library with oligos ZC37967 (SEQ ID NO:24) and ZC37972 (SEQ IDNO:25) with BamHI and Bgl2 restriction sites engineered at the 5′ and 3′ends, respectively, under conditions as follows: 25 cycles of 94° C. for60 sec., 57° C. for 60 sec., and 72° C. for 120 sec.; and 72° C. for 7min. PCR products were purified using QIAquick PCR Purification Kit(Qiagen), digested with BamHI and Bgl2 (Boerhinger-Mannheim), separatedby gel electrophoresis and purified using a QIAquick gel extraction kit(Qiagen). The hzcytor19 BamHI/Bgl2 fragment was ligated into Fc4/pzmp20vector that had been digested with Bgl2. The zcytor19 fragment is clonedbetween a tPA leader peptide and human Fc4 fragment. Once the sequenceis confirmed, the DNA fragement of zcytor19 with tPA leader peptide wascut out by EcoRI, and Bgl2 diestion, and then cloned intopzp9/zcytor7/Fc4-CEE vector. This vector has the extracellular portionof hzcytor7 fused to Fc4 with a CEE tag, and digesting with EcoRI andBamHI removes the extracellular portion of hzcytor7 and allowssubstitution of hzcytor19. Minipreps of the resulting ligation werescreened for an EcoRI/BamHI insert of the correct size and positiveminipreps were sequenced to confirm accuracy of the PCR reaction.

For construction of the hCRF2-4/Fc4-cHIS portion of the heterodimer, theextracellular portion of hCRF2-4 was amplified by PCR from pZP-9 CRFwith oligos ZC39319 (SEQ ID NO:68) and ZC39325 (SEQ ID NO:70) underconditions as follows: 30 cycles of 94° C. for 60 sec., 57° C. for 60sec., and 72° C. for 120 sec; and 72° C. for 7 min. PCR product werepurified as described above and then digested with EcoRI and BamHI.Because the PCR product had an internal EcoRI site two bands wereobtained upon digestion: a 0.101 kB EcoRI/EcoRI fragment and a 0.574 kBEcoRI/BamHI fragment. The 0.574 EcoRI/BamHI fragment was ligated intovector#249 pHZ-1 DR1/Fc4-TCS-cHIS that had been digested with EcoRI andBamHI. This vector has the extracellular portion of hDR-1 fused to Fc4with a C-HIS tag (SEQ ID NO:[#]), and digesting with EcoRI and BamHIremoves the extracellular portion of hDR-1 and allows substitution ofhCRF2-4. Minipreps of the resulting ligation were screened for anEcoRI/BamHI insert of the correct size, and positive minipreps, wereEcoRI digested and band purified for further construction. The 0.101 kBEcoRI/EcoRI fragment was ligated into the EcoRI digested minipreps andclones were screened for proper orientation of insertion by KpnI/NdeIrestriction digestion. Clones with the correct size insertion weresubmitted for DNA sequencing to confirm the accuracy of the PCRreaction.

About 16 μg each of the hzcytor19/Fc4-cEE and hCRF2-4/Fc-4-cHIS wereco-transfected into BHK-570 (ATCC No. CRL-10314) cells usingLipofectamine (Gibco/BRL), as per manufacturer's instructions. Thetransfected cells were selected for 10 days in DMEM 5% FBS (Gibco/BRL)containing 1 μM methotrexate (MTX) (Sigma, St. Louis, Mo.) and 0.5 mg/mlG418 (Gibco/BRL) for 10 days. The resulting pool of transfectants wasselected again in 10 μM MTX and 0.5 mg./ml G418 for 10 days.

Example 24 Purification of Zcytor19/CRF2-4 Heterodimer Receptor

Conditioned culture media zcytor19/CRF2-4 heterodimer was filteredthrough 0.2 μm filter and 0.02% (w/v) Sodium Azide was added. Theconditioned media was directly loaded a Poros Protein A 50 Column at10-20 ml/min. Following load the column was washed with PBS and thebound protein eluted with 0.1M Glycine pH 3.0. The eluted fractionscontaining protein were adjusted to pH 7.2 and Concentrated to <80 mlusing YM30 Stirred Cell Membrane (Millipore).

The 80 ml eluate from the Protein A column was loaded onto a 318 mlSuperdex 200 HiLoad 26/60 Column (Pharmacia). The column was eluted withPBS pH 7.2 at 3 ml/min. Protein containing fractions were pooled toeliminate aggregates. The Superdex 200 pool was adjusted to 0.5M NaCl,10 mM Imidazole using solid NaCl and Imidazole and the pH was adjustedto 7.5 with NaOH. The adjusted protein solution was loaded onto a 200 mlNiNTA column (Qiagen) at 2 CV/hr. The bound protein was eluted,following PBS wash of the column, with five concentration steps ofImidazole: 40 mM, 100 mM, 150 mM, 250 mM, 500 mM. The fractions elutedat each step of imidizole were pooled and analyzed by N-terminalsequencing. Pools containing heterodimer, determined by sequencing werepooled and concentrated to 50 ml using a YM30 Stirred Cell Membrane(Millipore). The 50 ml eluate from the NiNTA column was loaded onto a318 ml Superdex 200 HiLoad 26/60 Column (Pharmacia). The column waseluted with PBS pH 7.2 at 3 ml/min. Protein containing fractions werepooled to eliminate aggregates, as determined by SEC MALS analysis.

Purified proteins were analyzed by N-terminal sequencing, amino acidanalysis, and SEC-MALS. Binding affinities to its ligand (zcyto20, 21,22, 24, and 25) and biological activities including its neutralizingactivity were determined.

Example 25 IL28RA mRNA Expression in Liver and Lymphocyte Subsets

In order to further examine the mRNA distribution for IL28RA,semi-quantitative RT-PCR was performed using the SDS 7900HT system(Applied Biosystems, CA). One-step RT-PCR was performed using 100 ngtotal RNA for each sample and gene-specific primers. A standard curvewas generated for each primer set using Bjab RNA and all sample valueswere normalized to HPRT. The normalized results are summarized in Tables17-19. The normalized values for IFNAR2 and CRF2-4 are also shown.

TABLE 17 B and T cells express significant levels of IL28RA mRNA. Lowlevels are seen in dendritic cells and most monocytes. Cell/TissueIl28RA IFNAR2 CRF2-4 Dendritic Cells unstim .04 5.9 9.8 DendriticCells + IFNg .07 3.6 4.3 Dendritic Cells .16 7.85 3.9 CD14+ stim'd withLPS/IFNg .13 12 27 CD14+ monocytes resting .12 11 15.4 Hu CD14+ Unact.4.2 TBD TBD Hu CD14+ 1 ug/ml LPS act. 2.3 TBD TBD H. Inflamed tonsil 312.4 9.5 H. B-cells + PMA/Iono 4 & 24 hrs 3.6 1.3 1.4 Hu CD19+ resting6.2 TBD TBD Hu CD19+ 4 hr. PMA/Iono 10.6 TBD TBD Hu CD19+ 24 hr Act.PMA/Iono 3.7 TBD TBD IgD+ B-cells 6.47 13.15 6.42 IgM+ B-cells 9.06 15.42.18 IgD− B-cells 5.66 2.86 6.76 NKCells + PMA/Iono 0 6.7 2.9 Hu CD3+Unactivated 2.1 TBD TBD CD4+ resting .9 8.5 29.1 CD4+ Unstim 18 hrs 1.68.4 13.2 CD4+ + Poly I/C 2.2 4.5 5.1 CD4+ + PMA/Iono .3 1.8 .9 CD3 negresting 1.6 7.3 46 CD3 neg unstim 18 hrs 2.4 13.2 16.8 CD3 neg + PolyI/C 18 hrs 5.7 7 30.2 CD3 neg + LPS 18 hrs 3.1 11.9 28.2 CD8+ unstim 18hrs 1.8 4.9 13.1 CD8+ stim'd with PMA/Ion 18 hrs .3 .6 1.1

As shown in Table 18, normal liver tissue and liver derived cell linesdisplay substantial levels of IL28RA and CRF2-4 mRNA.

TABLE 18 Cell/Tissue IL28RA IFNAR2 CRF2-4 HepG2 1.6 3.56 2.1 HepG2 UGAR5/10/02 1.1 1.2 2.7 HepG2, CGAT HKES081501C 4.3 2.1 6 HuH7 5/10/02 1.6316 2 HuH7 hepatoma - CGAT 4.2 7.2 3.1 Liver, normal - CGAT #HXYZ020801K11.7 3.2 8.4 Liver, NAT—Normal adjacent tissue 4.5 4.9 7.7 Liver,NAT—Normal adjacent tissue 2.2 6.3 10.4 Hep SMVC hep vein 0 1.4 6.5 HepSMCA hep. Artery 0 2.1 7.5 Hep. Fibro 0 2.9 6.2 Hep. Ca. 3.8 2.9 5.8Adenoca liver 8.3 4.2 10.5 SK-Hep-1 adenoca. Liver .1 1.3 2.5 AsPC-1 Hu.Pancreatic adenocarc. .7 .8 1.3 Hu. Hep. Stellate cells .025 4.4 9.7

As shown in Table 19, primary airway epithelial cells contain abundantlevels of IL28RA and CRF2-4.

TABLE 19 Cell/Tissue IL28RA IFNAR2 CRF2-4 U87MG - glioma 0 .66 .99 NHBEunstim 1.9 1.7 8.8 NHBE + TNF-alpha 2.2 5.7 4.6 NHBE + poly I/C 1.8 ndnd Small Airway Epithelial Cells 3.9 3.3 27.8 NHLF—Normal human lungfibroblasts 0 nd nd

As shown in Table 20, ZcytoR19 is present in normal and diseased liverspecimens, with increased expression in tissue from Hepatitis C andHepatitis B infected specimens.

TABLE 20 Cell/Tissue IL28RA CRF2-4 IFNAR2 Liver with CoagulationNecrosis 8.87 15.12 1.72 Liver with Autoimmune Hepatitis 6.46 8.90 3.07Neonatal Hepatitis 6.29 12.46 6.16 Endstage Liver disease 4.79 17.0510.58 Fulminant Liver Failure 1.90 14.20 7.69 Fulminant Liver failure2.52 11.25 8.84 Cirrhosis, primary biliary 4.64 12.03 3.62 CirrhosisAlcoholic (Laennec's) 4.17 8.30 4.14 Cirrhosis, Cryptogenic 4.84 7.135.06 Hepatitis C+, with cirrhosis 3.64 7.99 6.62 Hepatitis C+ 6.32 11.297.43 Fulminant hepatitis secondary to Hep A 8.94 21.63 8.48 Hepatitis C+7.69 15.88 8.05 Hepatitis B+ 1.61 12.79 6.93 Normal Liver 8.76 5.42 3.78Normal Liver 1.46 4.13 4.83 Liver NAT 3.61 5.43 6.42 Liver NAT 1.9710.37 6.31 Hu Fetal Liver 1.07 4.87 3.98 Hepatocellular Carcinoma 3.583.80 3.22 Adenocarcinoma Liver 8.30 10.48 4.17 hep. SMVC, hep. Vein 0.006.46 1.45 Hep SMCA hep. Artery 0.00 7.55 2.10 Hep. Fibroblast 0.00 6.202.94 HuH7 hepatoma 4.20 3.05 7.24 HepG2 Hepatocellular carcinoma 3.405.98 2.11 SK-Hep-1 adenocar. Liver 0.03 2.53 1.30 HepG2 Unstim 2.06 2.982.28 HepG2 + zcyto21 2.28 3.01 2.53 HepG2 + IFNa 2.61 3.05 3.00 NormalFemale Liver - degraded 1.38 6.45 4.57 Normal Liver - degraded 1.93 4.996.25 Normal Liver - degraded 2.41 2.32 2.75 Disease Liver - degraded2.33 3.00 6.04 Primary Hepatocytes from Clonetics 9.13 7.97 13.30

As shown in Tables 21-25, ZcytoR19 is detectable in normal B cells, Blymphoma cell lines, T cells, T lymphoma cell lines (Jurkat), normal andtransformed lymphocytes (B cells and T cells) and normal humanmonocytes.

TABLE 21 HPRT IL28RA IL28RA IFNR2 CRF2-4 Mean Mean norm IFNAR2 normCRF2-4 Norm CD14+ 24 hr unstim #A38 13.1 68.9 5.2 92.3 7.0 199.8 15.2CD14+ 24 hr stim #A38 6.9 7.6 1.1 219.5 31.8 276.6 40.1 CD14+ 24 hrunstim #A112 17.5 40.6 2.3 163.8 9.4 239.7 13.7 CD14+ 24 hr stim #A11211.8 6.4 0.5 264.6 22.4 266.9 22.6 CD14+ rest #X 32.0 164.2 5.1 1279.739.9 699.9 21.8 CD14+ + LPS #X 21.4 40.8 1.9 338.2 15.8 518.0 24.2 CD14+24 hr unstim #A39 26.3 86.8 3.3 297.4 11.3 480.6 18.3 CD14+ 24 hr stim#A39 16.6 12.5 0.8 210.0 12.7 406.4 24.5 HL60 Resting 161.2 0.2 0.0214.2 1.3 264.0 1.6 HL60 + PMA 23.6 2.8 0.1 372.5 15.8 397.5 16.8 U937Resting 246.7 0.0 0.0 449.4 1.8 362.5 1.5 U937 + PMA 222.7 0.0 0.0 379.21.7 475.9 2.1 Jurkat Resting 241.7 103.0 0.4 327.7 1.4 36.1 0.1 JurkatActivated 130.7 143.2 1.1 Colo205 88.8 43.5 0.5 HT-29 26.5 30.5 1.2

TABLE 22 HPRT SD IL28RA SD Mono 24 hr unstim #A38 0.6 2.4 Mono 24 hrstim #A38 0.7 0.2 Mono 24 hr unstim #A112 2.0 0.7 Mono 24 hr stim #A1120.3 0.1 Mono rest #X 5.7 2.2 Mono + LPS #X 0.5 1.0 Mono 24 hr unstim#A39 0.7 0.8 Mono 24 hr stim #A39 0.1 0.7 HL60 Resting 19.7 0.1 HL60 +PMA 0.7 0.4 U937 Resting 7.4 0.0 U937 + PMA 7.1 0.0 Jurkat Resting 3.71.1 Jurkat Activated 2.4 1.8 Colo205 1.9 0.7 HT-29 2.3 1.7

TABLE 23 Mean Mean Mean Mean Hprt IFNAR2 IL28RA CRF CD3+/CD4+ 0 10.185.9 9.0 294.6 CD4/CD3+ Unstim 18 hrs 12.9 108.7 20.3 170.4 CD4+/CD3+ +Poly I/C 18 hrs 24.1 108.5 52.1 121.8 CD4+/CD3+ + PMA/Iono 18 hrs 47.883.7 16.5 40.8 CD3 neg 0 15.4 111.7 24.8 706.1 CD3 neg unstim 18 hrs15.7 206.6 37.5 263.0 CD3 neg + Poly I/C 18 hrs 9.6 67.0 54.7 289.5 CD3neg + LPS 18 hrs 14.5 173.2 44.6 409.3 CD8+ Unstim. 18 hrs 6.1 29.7 11.179.9 CD8+ + PMA/Iono 18 hrs 78.4 47.6 26.1 85.5 12.8.1 - NHBE Unstim47.4 81.1 76.5 415.6 12.8.2 - NHBE + TNF-alpha 42.3 238.8 127.7 193.9SAEC 15.3 49.9 63.6 426.0

TABLE 24 IL28RA CRF IFNAR2 IL28RA CRF IFNAR2 Norm Norm Norm SD SD SDCD3+/CD4+ 0 0.9 29.1 8.5 0.1 1.6 0.4 CD4/CD3+ Unstim 18 hrs 1.6 13.2 8.40.2 1.6 1.4 CD4+/CD3+ + Poly I/C 18 hrs 2.2 5.1 4.5 0.1 0.3 0.5CD4+/CD3+ + PMA/Iono 18 hrs 0.3 0.9 1.8 0.0 0.1 0.3 CD3 neg 0 1.6 46.07.3 0.2 4.7 1.3 CD3 neg unstim 18 hrs 2.4 16.8 13.2 0.4 2.7 2.3 CD3neg + Poly I/C 18 hrs 5.7 30.2 7.0 0.3 1.7 0.8 CD3 neg + LPS 18 hrs 3.128.2 11.9 0.4 5.4 2.9 CD8+ Unstim. 18 hrs 1.8 13.1 4.9 0.1 1.1 0.3CD8+ + PMA/Iono 18 hrs 0.3 1.1 0.6 0.0 0.1 0.0 12.8.1 - NHBE Unstim 1.68.8 1.7 0.1 0.4 0.1 12.8.2 - NHBE + TNF-alpha 3.0 4.6 5.7 0.1 0.1 0.1SAEC 4.1 27.8 3.3 0.2 1.1 0.3

TABLE 25 SD SD SD SD Hprt IFNAR2 IL28RA CRF CD3+/CD4+ 0 0.3 3.5 0.6 12.8CD4/CD3+ Unstim 18 hrs 1.4 13.7 1.1 8.5 CD4+/CD3+ + Poly I/C 18 hrs 1.39.8 1.6 3.4 CD4+/CD3+ + PMA/Iono 18 hrs 4.0 10.3 0.7 3.7 CD3 neg 0 1.416.6 1.6 28.6 CD3 neg unstim 18 hrs 2.4 16.2 2.7 12.6 CD3 neg + Poly I/C18 hrs 0.5 7.0 1.0 8.3 CD3 neg + LPS 18 hrs 1.0 39.8 5.6 73.6 CD8+Unstim. 18 hrs 0.2 1.6 0.5 6.1 CD8+ + PMA/Iono 18 hrs 1.3 1.7 0.2 8.112.8.1 - NHBE Unstim 2.4 5.6 2.7 2.8 12.8.2 - NHBE + TNF-alpha 0.5 3.43.5 3.4 SAEC 0.5 4.8 1.8 9.9

Example 26

Inhibition of IIL28A, IL29, and Zcyto24 Signalling with the SolubleHeterodimer (zcytor19/CRF2-4), and with the Soluble Homodimer

Signal Transduction Reporter Assay

A signal transduction reporter assay can be used to show the inhibitorproperties of zcytor19-Fc4 homodimeric and zcytor19-Fc/CRF2-4-Fcheterodimeric soluble receptors on zcyto20, zcyto21 and zcyto24signaling. Human embryonal kidney (HEK) cells overexpressing thezcytor19 receptor are transfected with a reporter plasmid containing aninterferon-stimulated response element (ISRE) driving transcription of aluciferase reporter gene. Luciferase activity following stimulation oftransfected cells with ligands (including zcyto20 (SEQ ID NO:52),zcyto21 (SEQ ID NO:55), and zcyto24 (SEQ ID NO:60) reflects theinteraction of the ligand with soluble receptor.

Cell Transfections

293 HEK cells overexpressing zcytor19 were transfected as follows:700,000 293 cells/well (6 well plates) were plated approximately 18 hprior to transfection in 2 milliliters DMEM+10% fetal bovine serum. Perwell, 1 microgram pISRE-Luciferase DNA (Stratagene) and 1 microgrampIRES2-EGFP DNA (Clontech,) were added to 6 microliters Fugene 6 reagent(Roche Biochemicals) in a total of 100 microliters DMEM. Thistransfection mix was added 30 minutes later to the pre-plated 293 cells.Twenty-four hours later the transfected cells were removed from theplate using trypsin-EDTA and replated at approximately 25,000 cells/wellin 96 well microtiter plates. Approximately 18 h prior to ligandstimulation, media was changed to DMEM+0.5% FBS.

Signal Transduction Reporter Assays

The signal transduction reporter assays were done as follows: Followingan 18 h incubation at 37° C. in DMEM 0.5% FBS, transfected cells werestimulated with 10 ng/ml zcyto20, zcyto21 or zcyto24 and 10micrograms/ml of the following soluble receptors; human zcytor19-Fchomodimer, human zcytor19-Fc/human CRF2-4-Fc heterodimer, humanCRF2-4-Fc homodimer, murine zcytor19-Ig homodimer. Following a 4-hourincubation at 37° C., the cells were lysed, and the relative light units(RLU) were measured on a luminometer after addition of a luciferasesubstrate. The results obtained are shown as the percent inhibition ofligand-induced signaling in the presence of soluble receptor relative tothe signaling in the presence of PBS alone. Table 26 shows that thehuman zcytor19-Fc/human CRF2-4 heterodimeric soluble receptor is able toinhibit zcyto20, zcyto21 and zcyto24-induced signaling between 16 and45% of control. The human zcytor19-Fc homodimeric soluble receptor isalso able to inhibit zcyto21-induced signaling by 45%. No significanteffects were seen with huCRF2-4-Fc or muzcytor19-Ig homodimeric solublereceptors.

TABLE 26 Percent Inhibition of Ligand-induced Interferon StimulatedResponse Element (ISRE) Signaling by Soluble Receptors Huzcytor19-Fc/Huzcytor19- HuCRF2-4- Muzcytor19- Ligand huCRF2-4-Fc Fc Fc Ig Zcyto2016% 92% 80% 91% Zcyto21 16% 45% 79% 103%  Zcyto24 47% 90% 82% 89%

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1-16. (canceled)
 17. A cultured cell comprising an expression vectorcomprising the following operably linked elements: a) a firsttranscription promoter; a first DNA segment encoding a first receptorsubunit comprising amino acid residues 21-520 of SEQ ID NO:19; and afirst transcription terminator; and b) a second transcription promoter;a second DNA segment encoding a second receptor subunit comprising theamino acid residues of SEQ ID NO:64; and a second transcriptionterminator; wherein the first receptor subunit and the second receptorsubunit form a heterodimeric receptor capable of binding a polypeptidecomprising amino acid residues 20-200 of SEQ ID NO:55.
 18. The culturedcell of claim 17, wherein the cultured cell is prokaryotic.
 19. Thecultured cell of claim 17, wherein the cultured cell is eukaryotic. 20.A cultured cell comprising: a) a first expression vector comprising: i)a first transcription promoter; ii) a first DNA segment encoding a firstreceptor subunit comprising amino acid residues 21-520 of SEQ ID NO:19;and iii) a first transcription terminator; and b) a second expressionvector comprising: i) a second transcription promoter; ii) a second DNAsegment encoding a second receptor subunit comprising the amino acidsequence of SEQ ID NO:64; and iii) a second transcription terminator;and wherein the first receptor subunit and the second receptor subunitform a heterodimeric receptor capable of binding a polypeptidecomprising amino acid residues 20-200 of SEQ ID NO:55.
 21. The culturedcell of claim 20, wherein the cultured cell is prokaryotic.
 22. Thecultured cell of claim 20, wherein the cultured cell is eukaryotic.